Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates.In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequencedependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.Double-stranded RNA (dsRNA) is a hallmark of viral infections, and thus, it is not surprising that the immune system has evolved the capacity to recognize dsRNA and respond to it by mounting antiviral responses. In vertebrates, these innate antiviral responses rely in part on the recognition of dsRNA by Toll-like receptor 3 and by RNA-dependent protein kinase (32, 47). The consequences of dsRNA recognition include activation of the interferon system, initiation of apoptosis, and inhibition of cellular protein synthesis. From an evolutionary perspective, innate immune activation by dsRNA has long been thought to be exclusive to vertebrates. This view has been encouraged by the fact that genes encoding homologues of interferons, their receptors, and most of the prominent interferon-regulated genes are absent in fully sequenced invertebrate genomes (1, 7, 10, 11). Nevertheless, it is a reasonable expectation that invertebrates should have an innate immune system capable of recognizing dsRNA as a signature of viral infection. A previous study suggested such a capability by demonstrating that exposure of a marine shrimp to dsRNA induced innate antiviral immunity in a sequence-independent manner (36). The mechanisms underlying this phenomenon as well as its occurrence in other invertebrate taxa remain unknown, but it is clear that the recognition of dsRNA by another pathway, RNA interference (RNAi), is widely distributed among invertebrates and likely an important component of the invertebrate antiviral response.RNAi comprises a set of related cellular processes by which dsRNA molecules direct the suppression of gene expression based on sequence homology between the dsRNA trigger and the target gene. The specific mechanisms u...

Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNAmediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

ABSTRACT:In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 ± 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27°C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD 50 and LD 90 . The LD 50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 × 10 7 (Trial 1) and 1:6.667 × 10 7 (Trial 2). The LD 50 s of 2 different WSSV injection preparations were estimated at 1:4.444 × 10 6 and 1:4.505 × 10 6 . The LD 50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20°C and 1:1272 at 27°C. A second waterborne TSV inoculum challenge at 27°C produced an LD 50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens. KEY WORDS: TSV · WSSV · Bioassay · Shrimp · Litopenaeus vannameiResale or republication not permitted without written consent of the publisher

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.KEY WORDS: Penaeid · Shrimp · White spot virus · Disease · Native species · PCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 59: [179][180][181][182][183][184][185] 2004 shipped to South Carolina from a previously certified SPF (specific pathogen free) producer that had become contaminated. Since 1996, South Carolina has not had any further TSV epizootic events. In January of 1997, WSSV was identified at culture facilities in South Carolina.WSSV was first reported from Northeast Asia in 1992 and spread throughout the region during the 1990s causing devastating declines in farmed shrimp production (Flegel 1997). WSSV was identified in captive shrimp in South Carolina in 1997, and the available evidence suggested the infections were derived from the wild (Browdy & Holland 1998). WSSV is unique among shrimp viruses in that it infects a variety of crustaceans. WSSV-like genetic material has been found in samples from white shrimp, grass shrimp, fiddler crabs, blue crabs, and stone crabs in South Carolina (R.W.C. unpubl. data). These crustaceans potentially serve as reservoirs for WSSV, with the possibility of re-infecting wild as well as farmed shrimp populations. Archived DNA samples suggest a WSSVlike virus may have existed in the southeastern USA as early as 1988 (R.W.C. unpubl. data). At present, there are not sufficient data to determine whether the WSSV infections identified in captive white shrimp in South Carolina resulted from a recent introduction from Asia or from indigenous carriers.Risks of shrimp virus introduction are not limited to transfer of live animals for shrimp culture. Two other potentially important sources of shrimp viruses are carrier organisms in ship ballast water and frozen seafood products. Invasions of a wide range of nuisance organisms, including a number of crustaceans, have been linked to the release of ballast water (Carlton 1992). In many...

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