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With Shrimp as Big Business, Researchers Look to RNAi to Treat Disease Outbreaks

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As the consumption of shrimp in the United States has climbed in recent years, shrimp farming has become big business. Last year, the value of shrimp imported to the US was $3.7 billion, according to the US Department of Agriculture.

As such, the need for effective strategies for dealing with disease outbreaks in the crustaceans has become more urgent. Yet for two of the biggest threats to farmed shrimp, Taura syndrome and white spot disease, there are no treatments currently available. However, a solution may be found in RNAi, according to a pair of researchers from the Medical University of South Carolina and their colleagues from the South Carolina Department of Natural Resources.

White spot disease is a fatal condition in shrimp caused by a double-stranded DNA virus and affects all decapod crustaceans from marine, brackish, and freshwater sources such as shrimp, lobsters, and crayfish. The disease was first reported in Taiwan and China around 1991, according to the US Department of Agriculture, and was discovered in the US in 1995. Since then, it has also been identified throughout Central and South America.

Taura syndrome is caused by the Taura syndrome virus, a single-stranded RNA virus in the Picornaviridae family. The disease was first described in Ecuador in 1992, and since then has been identified throughout Central and South America, as well as in the US and Mexico, the USDA states. Mortality in shrimp associated with the disease can range from 5 to 95 percent.

"Probably the biggest problem that shrimp farms face in terms of production is viral pathogens," Craig Browdy, a marine scientist at the South Carolina Department of Natural Resources, told RNAi News last week. "In fact, the Taura syndrome virus caused hundreds of millions of dollars in damage to the Ecuadorian shrimp industry in the mid-1990s. Around the late 1990s, the white spot syndrome virus really started spreading to many places around the world," he noted. "It's causing many billions of dollars in damages to the shrimp industry in places like Thailand [and] it really crippled the industry in India."

Browdy added that although shrimp farmers have developed husbandry and exclusion methods for dealing with outbreaks, the diseases continue to be problematic since no treatment exists.

With this in mind, Javier Robalino, a graduate student in the lab of MUSC researcher Greg Warr, began a series of experiments to see if clues to treating shrimp disease could be gleaned by using long pieces of double-stranded RNA to down-regulate endogenous shrimp genes implicated in antiviral immunity.

"I was born in Ecuador, and the shrimp farming industry is a big deal there," Robalino told RNAi News.

After studying marine biology and aquaculture as an undergraduate at Escuela Politecnica del Litoral, "I got into a scholarship program from the Ecuadorian government to [pursue] graduate studies in several areas of interest to the country, including marine biotechnology," he said.

This program brought Robalino to Warr's lab, where his colleagues were forming a marine genomics program. "I, Greg, and the others here were interested in possible mechanisms of antiviral immunity," Robalino said. But "when we started this project, there was literally absolutely nothing known about possible mechanism of antiviral immunity, not only in shrimp but in any invertebrate … including Drosophila."

By this time, reports of RNAi as a natural antiviral mechanism in animals had begun appearing, he said, so he and his colleagues began to investigate whether dsRNA could be used to examine possible components of signal transduction pathways that might play a role in viral resistance.

"We first demonstrated that we could knock down genes in vivo in shrimp [in a manner] similar to what [is done] in Drosophila — you inject naked long double-stranded RNA, and you get depletion of messenger RNA," Robalino explained. "Then we decided to put two and two together and ask, 'Can resistance or susceptibility to a specific viral challenge change when we knock down a shrimp gene?'."

According to Warr, "What [Robalino] discovered was that any long double-stranded RNA, in a sequence non-specific manner, would induce an innate antiviral immune response which was active against … white spot syndrome virus … and Taura virus. You could even use synthetic analogs of double-stranded RNA."

Robalino noted that it is currently unclear if this immune response is specific to certain diseases such as white spot disease and Taura syndrome, but that the question is being investigated at MUSC.

Robalino's discovery "was of great interest from a basic scientific discovery because it was the first time that double-stranded RNA had been shown to [elicit] … an innate antiviral response in an invertebrate," Warr told RNAi News. "The other interesting thing about this non-specific response … was that it could be overcome by much larger doses of challenge virus. For example, if you titrated the challenge dose of virus such that it would kill around 70 or 80 percent of the shrimp, then you could show with double-stranded RNA non-specific protection of true biological significance. However, if you increase the challenge dose … by a hundred- or a thousand-fold, you could essentially kill the shrimp as if they were not protected at all," he said.

This work was published in the Journal of Virology in October 2004.

Warr said that the fact that the dsRNA-triggered immune response could be overcome prompted Robalino to speculate that "if you coupled this with specific sequence effects, potentially RNAi, you would get a much more potent [immune] response."

According to Robalino, "We knew that if we got enough virus to a population of shrimp, it would be impossible to protect them from the infection using non-specific double-stranded RNA, so we started replacing that with [dsRNA targeting] just genes derived from the genomes of these viruses that just make sense as being essential for the viral lifecycle. So we tried structural proteins [and] envelope proteins, as well as the typical DNA polymerase genes and what not," he said.

"We saw a fairly general phenomenon [wherein] you can essentially block the pathogenesis of the virus [by] targeting many of the virally encoded genes," Robalino said, noting that he and his colleagues were typically targeting a single gene at a time. "We used white spot and TSV to further show the sequence-specificity of these phenomena — so, no protection across viral phylogenies."

Robalino said that these findings have been submitted for publication in a peer-reviewed journal.

Despite the promise of RNAi as a method of treating disease in shrimp, Robalino and his colleagues are facing the same problem that everyone working to turn the technology into a therapeutic modality faces: delivery.

In their experiments, Robalino and his colleagues injected the dsRNA intramuscularly into individual shrimp. "Our major obstacle right now is to find a delivery system that will allow [RNAi] to be utilized in farm situations to inoculate billions of offspring at once," Bob Chapman, a marine scientist with the South Carolina Department of Natural Resources, said. "It also appears that the effect needs to be reinforced periodically, and how often that is we're not sure."

The researchers declined to comment on delivery approaches they are investigating, but a recently published patent application they filed — number 20050080032 — covering dsRNA-induced immunity in crustaceans indicates that they are exploring injection, immersion, and feeding methods.

The researchers are also interested in working with outside parties to advance their work. "We're very interested in potential partners with expertise in delivery systems," Warr said.

— DM

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