RNA INTERFERENCE-BASED ORAL THERAPEUTANT FOR WHITE SPOT SYNDROME VIRUS IN SHRIMP

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 0228809
• Grant No.: 2012-33610-19536
• Proposal No.: 2012-00262
• Project Start Date: Jul 1, 2012
• Project End Date: Feb 28, 2013
• Grant Year: 2012
• Program Code: [8.7]- Aquaculture

Recipient Organization
LOGOS ENERGY INC
51 ALPHA PARK
HIGHLAND HEIGHTS,OH 44143

Performing Department
(N/A)

Non Technical Summary
Aquaculture is the fastest growing animal food-producing sector and has been projected to play a very important role in meeting the food demand of the growing world population. Shrimp is the most important aquaculture commodity accounting for nearly twenty percent of the total value of international trade of fish products. However, as with other aquaculture species, shrimp aquaculture suffers significant losses (about 15 to 30 percent) annually due to disease outbreaks. White Spot disease (WSD), caused by the White Spot Syndrome Virus (WSSV), has been the most problematic viral pathogen affecting global shrimp farming since emerging in 1992. It is one of the most widespread viruses in the industry, occurring in virtually all shrimp farming countries. Over 50 crustacean species have been found to be susceptible to WSSV, including crayfish, crabs, lobsters, etc., which also act as carriers of the virus. It is highly virulent and may result in 80-100 percent mortality in ponds within 7-10 days of infection. Such extreme losses affect food security and cause profound socio-economic distress. Various strategies have been tried to control WSSV infection in shrimp - all with mixed results. In addition, various preventive strategies such as use of pathogen free shrimp larvae, application of bio-secure farming/aquaculture techniques, disinfection and use of WSSV resistant strains are commonly adopted to reduce the appearance of the pathogen. However, these strategies do not provide an effective protection once the viral infection appears in a shrimp farm. Currently, no viable commercial product exists to combat WSSV in shrimp. There is a clear and immediate need to develop a novel therapeutant and delivery method that can provide effective protection against WSSV in shrimp. RNA interference (RNAi) is a process within living cells that moderates the activity of their genes. RNAi based strategies appear to be promising in controlling WSSV in shrimp. Preliminary data suggest strongly that our RNAi strategy which targets four WSSV genes may provide very potent protection to shrimp against WSSV. In collaboration with an independent industry leading third party laboratory, an injection trial was conducted on juvenile shrimp, the results of which confirmed strongly the efficacy of our RNAi strategy in providing protection to shrimp against WSSV. It is anticipated that the work proposed in this project will produce new fundamental and applied knowledge that will be shared via collaboration, publications, conferences and word-of-mouth. Success in this project will catalyze action to commercialize a much needed product and validate methods to control viral disease that could open up a new area for improved disease control in animal husbandry. This project proposes the development of an RNAi-based therapeutant against WSSV of shrimp delivered orally through microalgae/yeast consumed by the shrimp.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3721	1040	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3721 - Marine shrimp;

Field Of Science
1040 - Molecular biology;

Keywords

white spot syndrome virus

wssv

wsd

microalgae

yeast

shrimp

therapeutant

rnai

oral

Goals / Objectives
1) Development of transgenic algal/yeast strains expressing dsRNA targeting WSSV genes. 2)Test whether feeding of transgenic algae/yeast expressing dsRNA targeting WSSV genes to shrimp provides protection against WSSV. 3) Quantitate the amount of dsRNA/siRNA produced by transgenic algal/yeast clones found to be effective in controlling WSSV infection in shrimp.

Project Methods
Approach: For this work four WSSV gene targets have been selected based on published work that demonstrated that injection of dsRNA targeting these genes into shrimp provides protection against WSSV. A stretch of 250 to 300 nucleotides, not showing significant homology to any human, animal or insect sequences, will be selected from the WSSV target genes. The sequences will be amplified by PCR from WSSV genomic DNA for cloning. Gene sequences from pairs of these genes will be used to create two inverted repeat (IR) constructs. The fused IR's will be cloned into transformation vectors under the control of a strong constitutive promoter. Nuclear and chloroplast transformation will be performed with the IR's constructs following the standard transformation protocols for the host organisms. Transgenic clones will be selected based on their ability to grow on selective media. Transgenic clones will be analyzed by PCR to confirm integration of the IR's into the genome. Feeding trials will be carried out using transgenic algae and yeast expressing dsRNA targeting WSSV genes to check their efficacy in controlling WSSV infection. All the trials will be carried out in a biosecure and biocontained facility. Transgenic algae/yeast biomass will be incorporated into cold extruded diet. Feeding trial will be carried out in replicates on a minimum of 15 shrimps, 0.5 to 1 g (approximately PL 45) per treatment. All the data will be analyzed for statistical significance. At the end of the trial all dead / live shrimp will be tested for presence of WSSV by PCR. The existence of any correlation between the level of protection provided by a transgenic algal/yeast clone and the level of dsRNA/siRNA targeting the WSSV target genes produced by that clone will also be investigated. A positive correlation would help in identifying the promising clones for trials before conducting an actual feeding trial. This would significantly reduce the cost and the time required to develop a similar product. The Ambion mirVana miRNA detection kit will be used to quantitate the amount of siRNA's targeting WSSV genes present in the total cellular RNA from transgenic algal/yeast clones following the manufacturer's protocol. The Ambion mirVana miRNA detection kit is very sensitive and can detect as little as 10 attomoles of target RNA and is especially designed for detection of siRNA. Known amounts of synthetic siRNAs will be used along with the samples to quantitate the amount of siRNAs produced by transgenic algal/yeast clones. Outcome: The above mentioned experiments will provide information whether delivery of ds/siRNA targeting WSSV genes through microalgae/yeast can protect shrimp from WSSV infection.

Progress 07/01/12 to 02/28/13

Outputs
OUTPUTS: Following activities were conducted to meet the objectives of this project: 1. Production of plasmid constructs for expression of double stranded RNA (dsRNA) targeting the white spot syndrome virus (WSSV) genes from Chlamydomonas nuclear genome. 2. Production of Chlamydomonas nuclear transformants expressing dsRNA targeting WSSV genes. 3. Production of plasmid constructs for expression of dsRNA targeting WSSV genes from yeast. 4. Production of transgenic yeast strains expressing dsRNA targeting the WSSV genes analogous to those for algae described above. 5. Feeding trials to test whether oral delivery of RNA interference (RNAi) initiating elements via feed supplemented with transgenic algae or yeast expressing dsRNA targeting WSSV genes is effective in protecting shrimp against WSSV infection. 6. Viral challenge trials were carried out on shrimp fed experimental and control diets designed to provide protection against WSSV in collaboration with the University of Arizona. 7.A high school student intern from the Clyde C. Miller Career Academy, Saint Louis, Missouri was trained during the course of this project in techniques related to algal cultivation and harvesting Dissemination of results: The results of the research work have not been shared with any entity at this point of time because the product is in early stages of development. PARTICIPANTS: Dr. Anil Kumar: Dr. Kumar worked as the Principal Investigator on this project. He was responsible for experimental design, data collection/analysis, coordination and execution of all experimentation at the Logos Energy laboratory in Saint Louis, MO. In addition, Dr. Kumar was involved in planning and coordination of shrimp feeding trials conducted at University of Arizona. University of Arizona (UAZ): UAZ worked as a partner organization/collaborator for this project. The feeding trials on shrimp were carried out at UAZ in Dr. Donald Lightner's laboratory. Dr. Donald Lightner is one of the world's leading experts on shrimp aquaculture and shrimp viral pathogens. Phycal Inc.: Phycal Inc. is a subsidiary of Logos Energy. Phycal worked as a partner organization. Production of yeast biomass for shrimp feeding trials was carried out at Phycal's Cleveland facility. Dr. F. C. Thomas Allnutt: Dr. Allnutt is Senior Vice President, Research at Phycal. Dr. Allnutt's experience and expertise in the feed industry was utilized in planning shrimp feeding trials and formulating feeds for the feeding trials. Dr. Allnutt was also involved in production of yeast biomass for the shrimp feeding trials at Phycal's Cleveland facility. Dr. Richard Sayre: Dr. Sayre is Chief Technology Officer at Phycal. Dr. Sayre volunteered to provide scientific oversight and mentoring to all personnel involved in development of transgenic microalgal and yeast strains relevant to this project. Mr. Ken Moats: Mr. Moats worked with Dr. Allnutt for the production of yeast biomass for this project. Mr. Alexander Perwich: Mr. Perwich serves as President of New Business Development at Logos Energy. Mr. Perwich volunteered to coordinate all the communication between USDA and Logos Energy for this project. Ms. Markeeta Gant: Ms. Gant is a high school student at the Clyde C. Miller Career Academy, Saint Louis, Missouri. Ms. Gant volunteered to work as an intern on this project. Ms. Gant was trained during the course of this project in techniques related to algal cultivation and harvesting. TARGET AUDIENCES: The target audience for this project is aquaculture and aquaculture feed industry. There is a strong need in the shrimp aquaculture industry for effective control of viral diseases. If successful, the RNAi based oral therapeutic development approach used in this work can revolutionize application of preventative treatment in aquaculture. However, since the product is in early stages of development, we have not yet shared the results of the research work with the target audience. But we do intend to share the results of this work with the target audience through publication of a research report or article following some more experiments intended to improve stability and delivery of RNAi elements to shrimp PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The resources provided during this project were used to develop transgenic algae and yeast strains expressing dsRNAs targeting WSSV genes and conducting feeding trials with shrimp. The feeding trials on shrimp were carried out in collaboration with Dr. Donald Lightner at University of Arizona. The objective of this project was to develop an RNAi based oral therapeutic against WSSV of shrimp. A change in knowledge was discovered in the feeding trials conducted so far, in that no observed effective protection to shrimp against WSSV through delivery of ds/siRNA targeting WSSV genes through feed supplemented with transgenic algae and yeast expressing dsRNA targeting WSSV genes. There can be many different reasons why the oral delivery of transgenic algae/yeast expressing dsRNA targeting WSSV genes was not effective in protecting shrimp against WSSV. This could be due to: 1.Degradation of ds/siRNA during feed preparation. 2.Degradation of ds/siRNA during feed delivery as the feed is dropped in a water tank. 3.Degradation of ds/siRNA inside shrimp gut. 4.Delivery of insufficient amount of ds/siRNA via feed to trigger the RNAi pathway. 5.Use of significantly higher inoculums of WSSV during feeding trials. An effective RNAi based oral therapeutic can only be developed after resolving the above mentioned issues associated with oral delivery of RNAi elements. It is widely acknowledged in the scientific community that development of strategies to deliver RNAi elements to the target organism or tissue is the key to development of RNAi based therapeutics. Therefore, a change in action was needed in order to stabilize the ds/siRNA expressed in these microbes and the development of strategies that ensure delivery of intact ds/siRNA molecules to the target organism (shrimp in this case) before conducting more feeding trials. The transgenic microbes were stabilized with cross-linker to destroy nucleases and provide a more stable form of dsRNA to the shrimp. However, to this date, no effective control of viral infection has been observed.

Publications

• No publications reported this period