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Journal of Invertebrate Pathology 110 (2012) 261266

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Journal of Invertebrate Pathology


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Nucleic-acid based antivirals: Augmenting RNA interference to vaccinate Litopenaeus vannamei


Lyric C. Bartholomay a,, Duan S. Loy b, J. Dustin Loy c, D.L. Harris c,d,e
a

Iowa State University, Department of Entomology, 442 Science II, Ames, IA 50011, USA Iowa State University, Department of Veterinary Microbiology and Preventive Medicine, Ames, IA 50011, USA c Harrisvaccines, 1102 S. Hills Dr. Suite 101, Ames, IA 50010, USA d Iowa State University, Department of Animal Science, 11 Kildee Hall, Ames, IA 50011, USA e Iowa State University, Department of Veterinary Diagnostic and Production Animal Medicine, Ames, IA 50011, USA
b

a r t i c l e

i n f o

a b s t r a c t
The Pacic white shrimp, Litopenaeus vannamei (Penaeidae: Litopenaeus) has emerged as the dominant farmed shrimp species globally in tropical countries. Rearing animals at high density in semi-intensive or intensive culture systems, and translocating animals across the globe, have created optimum conditions for devastating epizootics. Of the various pathogens that impact shrimp culture, viruses are arguably the most important infectious disease agents that exact devastating economic losses to the industry. Augmenting the RNA interference (RNAi) capacity of shrimp is a promising, emerging solution to prevent disease caused by a variety of highly pathogenic shrimp viruses. Indeed RNAi functions as a primary mechanism of antiviral RNA in arthropods, as was revealed initially in studies of mosquito-virus interactions. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) can be used as RNAi triggers in vivo in L. vannamei to reduce the pathology associated with virus infection. We explored the efcacy of those triggers as a function of the target gene in the virus genome and show that efcacy is virus-specic and cannot be predicted based on the target gene function or transcript level in an infected cell. Further, we show that carefully designed RNAi triggers provide an immune stimulus that results in specic, longterm protection and therefore suggest that these dsRNA antivirals can function as vaccines in controlling disease. 2012 Elsevier Inc. All rights reserved.

Article history: Available online 10 March 2012 Keywords: RNA interference Litopenaeus vannamei WSSV IMNV Innate immunity

Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . RNA interference . . . . . . . . . . . . . Induced RNAi to mitigate shrimp Developing the vaccine approach Acknowledgments . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .......................... .......................... disease: a vaccine approach? . .......................... .......................... .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 262 264 265 265 265

1. Introduction Viral diseases that impact cultured shellsh are a considerable impediment to successful, sustainable aquaculture practices and trade. The Pacic white shrimp, Litopenaeus vannamei (Penaeidae: Litopenaeus) is uniquely amendable to culture and has emerged as the dominant farmed shrimp species globally in tropical countries
Corresponding author. Fax: +1 515 294 5957.
E-mail address: lyricb@iastate.edu (L.C. Bartholomay). 0022-2011/$ - see front matter 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.03.002

that provide optimal climate and habitat; for example, shrimp farming has almost completely shifted from native Penaeus monodon to exotic L. vannamei in Southeast Asia in just the last decade (FAO Fisheries and Aquaculture Department, 2010). This shift facilitated global crustacean production increases of 15% annually between 2000 and 2008 (FAO Fisheries and Aquaculture Department, 2010). Disease outbreaks have ensued or intensied with the widespread adoption of this species, which has a native range that extends from Mexico to Peru on the Pacic Coast of the Americas (Holthius, 1980); as of 2008, more than 80% of culture of L. vannamei

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occurs outside the Americas (FAO Fisheries and Aquaculture Department, 2010). Translocation of the animal and of pathogenic viruses therein, and the consequences of rearing exotic animals at high density in monoculture have created a perfect storm for devastating epizootics; a prime example is the spread and establishment of IMNV from Brazil (2002) to Indonesia (2006) which has caused thus far incalculable losses to the Indonesian shrimp industry (Lightner, 2011). Of the various pathogens that impact shrimp culture, viruses are arguably the most important infectious disease deterrents to the productivity of the industry. Viruses are well-represented (9/ 10) amongst the etiologic agents of crustacean diseases that infect penaeid shrimp and are under study or listed by the World Organization for Animal Health (OIE), which sets international policies for animal trade (see Lightner, 2011). A diversity of taxa and (therefore) genome types are represented amongst the viruses of shrimp, including enveloped and nonenveloped, single stranded (ss) and double stranded (ds) RNA and DNA viruses. The viruses that are listed by the OIE and impact L. vannamei are ssRNA viruses that include Taura syndrome (TSV: Dicistroviridae), Yellowhead (YHV: Roniviridae), a dsRNA virus, Infectious myonecrosis (IMNV: Totiviridae), a ssDNA virus, Penaeus stylirostris densovirus (PstDNV: Parvoviridae), and a dsDNA virus, white spot syndrome (WSSV: Nimaviridae). Collectively, outbreaks of these viruses have cost the industry billions of dollars (Walker and Winton, 2010). For an individual farmer, an outbreak of disease can result in complete loss of a crop that necessitated expenditures for stock animals, feed and months of labor inputs; further, the threat of economic losses caused by disease prevents attempts to raise multiple crops per annum in some geographic regions, due to seasonal variation in pond conditions and temperature (Walker and Winton, 2010). The capacity to control viral diseases is largely limited to pathogen exclusion via specic pathogen-free procurement and biosecurity measures (Lightner, 2005) as well as the use of selective breeding to produce pathogen-resistant stocks (Argue, 2002), which has limited success depending on the virus. Continued economic losses underscore the importance of developing practical and efcacious means of protecting shrimp against viral disease to offset these losses and facilitate more protable production of farmed shrimp.

2. RNA interference Augmenting the RNA interference (RNAi) response in shrimp to suppress virus infection and pathology is a promising, emerging approach to shrimp virus disease control (Robalino et al., 2005; Robalino et al., 2007; Krishnan et al., 2009; Shekhar and Lu, 2009; Hirono et al., 2010). RNAi is a subcellular process that employs elements of small RNA processing pathways (SRRPs) to recognize and bind dsRNA and subsequently degrade matching RNA sequences, thereby preventing gene expression in a sequence-specic manner. RNAi plays a critical role in the arthropod innate immune response to virus infection. Indeed perhaps the rst evidence of the antiviral capacity of RNAi in arthropods was observed in vitro, in mosquito cell culture. A Sindbis Virus (SINV) vector engineered to express an antisense segment of the La Crosse Virus (LACV) small genome segment was used to infect cells in vitro that then were super-infected with LACV. Cells infected with the SINVLACV yielded far less LACV than control cells, leading Powers et al. to postulate that this could be a form of intracellular immunization against arboviruses (Powers et al., 1994). This capacity was effective in suppressing LACV, Dengue (DENV) or Yellow fever virus (YFV) infection; cells transduced with an expression vector that produced a portion of one of these genomes were accordingly resistant to LACV, DENV or YFV (Gaines et al., 1996; Olson et al., 1996; Powers et al., 1996; Higgs et al., 1998). Notably, these

studies followed shortly after the rst descriptions of pathogen-derived resistance in transgenic plants mediated by expression of a non-coding region of the Tobacco etch virus genome (Lindbo and Dougherty, 1992). This also preceded the understanding that dsRNA triggers gene suppression in a sequence-specic manner in Caenorhabdidis elegans (Fire et al., 1998) and that dsRNA can be used to suppress genes in vivo in Drosophila melanogaster (Kennerdell and Carthew, 1998). Comprehensive work with DENV and engineered SINV clearly revealed that the orientation (sense or antisense) of the sequence did not affect virus suppression, strongly implicating RNAi as the mechanism underpinning the antiviral effect (Adelman et al., 2001). The molecular drivers for RNAi in Drosophila were subsequently characterized to include: Dicer (a type-III ribonuclease that breaks down dsRNA or micro RNA (miRNA) into 21 nt duplexes), and elements of the RNA silencing effector complexes (RISCs). The RISC contains Argonaute (Ago) (short RNA binding proteins), R2D2 (shuttling short RNA from Dicer 2 into RISC), and accessory proteins that target, bind, and breakdown specic mRNAs as reviewed recently (Kemp and Imler, 2009). To identify specic genes in the RNAi pathway that drive antiviral responses, Hoa et al. used dsRNA to suppress two Dicer and ve Ago genes and found that Dicer 2, Argonaute 2 (Ago-2) and Argonaute 3 (Ago-3) were key to the RNAi response in a mosquito (Anopheles) cell line (Hoa et al., 2003). In vivo, suppression of Ago-2 increased the titer and extent of dissemination of Onyong-nyong virus (Togaviridae) in the mosquito, Anopheles gambiae (Keene et al., 2004). This was conclusive evidence of the important role of RNAi in the innate immune response to virus infection. Subsequently, RNAi responses were shown to protect D. melanogaster from infection by several RNA viruses: Flock house virus (FHV: Nodaviridae), Cricket paralysis virus (CrPV: Dicistroviridae) (van Rij et al., 2006; Wang et al., 2006), Drosophila C virus (DCV: Dicistroviridae), and Drosophila X virus (DXV: Birnaviridae) (Zambon et al., 2005). As was observed in mosquitoes, the nuclease Ago-2 was shown to be essential for antiviral defense against both DCV and CrPV. Flies defective in Ago-2 expression showed a signicant increase in viral RNA accumulation, virus titer and mortality (van Rij et al., 2006). Also, an enhanced disease susceptibility to virus infection was observed in mutant ies defective in expression of Dicer 2 or R2D2 (Galiana-Arnoux et al., 2006; Wang et al., 2006). Therefore, in the current understanding of the antiviral RNAi response in Drosophila and mosquitoes (Blair, 2011; van Mierlo et al., 2011), Dicer-2/R2D2/Ago-2 are engaged in recognizing and processing a dsRNA trigger that is provided exogenously or produced as an intermediate in the process of virus replication. Germane to this review, Dicer-2 and Ago-2 have been characterized and function in the antiviral response in penaeid shrimp (Unajak et al., 2006; Dechklar et al., 2008; Su et al., 2008; Labreuche et al., 2010; Chen et al., 2011). Augmenting the RNAi response by providing exogenous virusspecic RNAi triggers to be processed by Dicer-2/R2D2/Argonaute 2, alters the arthropod host phenotype in the face of virus infection to a variety of ends and potential applications. In vector mosquitoes, a transgenic approach to RNAi-mediated resistance to virus infection is proposed as a means to develop population replacement strategies to control transmission of mosquito-borne viruses like DENV (Travanty et al., 2004; Franz et al., 2006). RNAi-mediated resistance has been proposed as a means to suppress disease caused by Chinese sacbrood virus in the Chinese honey bee, Apis cerana (Liu et al., 2010) and Israeli acute paralysis virus in the European honeybee A. mellifera (Maori et al., 2009) in an effort to control colony collapse disorder. The latter approach already is manifest in a product called Remebee-I that is delivered to bees orally and has been used in eld trials to test feasibility of large-scale implementation of RNAi for disease control in an arthropod (Hunter et al., 2010).

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Exploiting or augmenting the RNAi response using a virus-specic RNAi trigger also is attractive as a means to specically disable economically signicant viruses of shrimp and thereby prevent morbidity and mortality. There is ample evidence that specic RNAi triggers mitigate viral disease in shrimp caused by, for example, WSSV (Robalino et al., 2005; Xu et al., 2007), YHV (Tirasophon et al., 2005; Yodmuang et al., 2006; Tirasophon et al., 2007), PstDNV (formerly Infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Ho et al., 2011), TSV (Robalino et al., 2005) and IMNV (Loy et al., 2012). Given the gravity of the economic impact of endemic viral disease, and in the interest of preparedness for emerging disease outbreaks, strategic development of new RNAi triggers as antiviral molecules is of great interest. The design of dsRNA antivirals often begins with the use of standard algorithms for dsRNA design to account for RNA secondary structure and prevent off-target effects, such as suppressing a host gene (accounted for by subjecting potential target sites to BLAST analysis against available host genome and transcriptome sequence). We posit that this process should be informed also by careful selection of virus genome location to streamline the duration and cost of research and product development for extant and emerging shrimp virus infections. Indeed, this has proven the case with our own research on antiviral molecule development for IMNV (Loy et al., 2012). For this review, the RNAi trigger sequence, gene function and efcacy of the antiviral effect will be reviewed based on the literature in an effort to delineate any potential patterns that could inform future work on antiviral RNAi trigger development. The development of RNAi triggers as antivirals for disease prevention in penaeid shrimp has encompassed a variety of proteinencoding genes as targets, and this technology has proven effective in mitigating disease in L. vannamei, P. monodon, and Marsupenaeus japonicus. In many studies, RNAi triggers were designed to target a compilation of structural and non-structural gene targets. For

example, in P. monodon, dsRNA to PmDNV ns1 (non-structural 1) or vp genes (structural genes) suppressed virus replication, but the ns1 gene target was more effective (Attasart et al., 2011). The literature that represents in vivo trials of RNAi triggers in L. vannamei represents several viruses including YHV, IMNV, TSV, and PstDNV, with most emphasis on WSSV infections. We have summarized this work on L. vannamei in Table 1, which delineates the target, dose of RNAi trigger, and resulting survival rate (if available) for each study. The data in Table 1 reveal no particular predictability in target choice efcacy based on virus gene category (structural or non-structural), and do not necessarily support the idea put forth that non-structural targets are good targets for RNAi trigger development because these tend to have to less abundant transcript levels relative to structural genes (Ho et al., 2011). Several RNAi triggers based on structural protein coding regions in the WSSV genome appear to provide strong protection from disease. Indeed, RNAi triggers for structural genes such as WSSV VP19 or VP28 work well to inhibit WSSV replication (see Table 1), despite the fact that these transcripts are highest in abundance as compared to other WSSV genes according to microarray analysis (Marks et al., 2005). Furthermore, in our own experience, an RNAi trigger designed to target the non-structural RNA-dependent RNA polymerase (RdRp) of IMNV proved far less effective than an RNAi trigger to a capsid gene (Loy et al., 2012). Beyond the general virus gene type, in the rst paper to describe RNAi as a potential antiviral for shrimp, Robalino et al. noted that virus genes likely are differentially susceptible to RNAi, and/or that the efcacy of RNAi trigger design could be dictated by whether or not the gene target is required for virus pathogenicity (Robalino et al., 2005). Our work with IMNV tested and supports both of these possibilities. Through an iterative process of narrowing down the most effective target in the genome, we found that a predicted protease cleavage product, Protein 1 (93 aa), in ORF 1 frame 1 was

Table 1 RNAi trigger vaccine candidate molecules for viruses that infect cultured shrimp with emphasis on Litopenaeus vannamei. Viruses are presented according to genome type, and RNAi triggers are shown according to gene name, dose provided to animals and animal weight at vaccination, and efcacy as measured by survival rate post-vaccination. RNAi triggers that were composed of short interfering RNA (siRNA) are indicated accordingly. Virus RNA viruses YHV (+ssRNA) Target gene RNA-dependent RNA polymerase Protease GFP Protease RdRP Major capsid protein Protein 1 (unknown function) Protein 1 (truncated) DNA viruses WSSV (dsDNA) DNA polymerase siRNA WSSVRR2 siRNA Thymid-ine/-ylate kinase siRNA vp24 siRNA vp28 siRNA Mutant siRNA vp19 Dose/size 25 lg/1015 g 25 lg/5 g 5 lg/12 g 2 lg/35 g 2 lg/35 g 0.2 lg 2 lg/35 g 0.02 lg 2 lg/35 g 10 lg/67 g SR (%) 88 100 50 90% 5 62 80 82 80 93100 50 50 66 33 33 0 81 72 29 78 44 85 0 5 87 79 n/a n/a Ref. Saksmerprome et al. (2009) Yodmuang et al. (2006) Robalino et al. (2005) Loy et al. (2012)

TSV (+ssRNA) IMNV (dsRNA)

Wu et al. (2007)

12 lg/12 g 1 lg 0.1 lg

Robalino et al. (2005)

WSSVRR2 WSSVDP vp28 vp19 siRNA Duck IgU vp28 vp26 ORF 1-2 overlap ORF 3

2 lg/12 g 4 lg 2.5 lg/25 g (3)

Mejia-Ruiz et al. (2011) Ho et al. (2011)

PstDNV (IHHNV)

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the most effective region of the genome to target for disease protection. Protein 1 shares sequence homology with previously described dsRNA binding proteins (Poulos et al., 2006; Nibert, 2007). This sequence is at the 5 most end of the IMNV genome, so it could be an effective RNAi trigger based on accessibility to the SRRP machinery, but could also be a powerful target if its function is in suppressing the RNAi response, thereby serving a critical role in virus pathogenicity (Loy et al., 2012). RNAi trigger development also can be impacted by the trigger length as a function of design. Several groups have designed short interfering RNAs (siRNAs), 21 bp in length, against virus targets with more limited success in disease protection. Robalino et al. (2005) noted that siRNAs are poor inducers of sequence-specic antiviral immunity and are incapable of mediating antiviral protection. For example, a full-length vp19 dsRNA provided high level protection, but an siRNA for the same target was not protective (Table 1). Further, an siRNA for the ribonucleotide reductase small subunit (rr2) provided 50% protection from WSSV disease (Xu et al., 2007), but a dsRNA provided 88% protection in a study by Robalino et al. (2005). In WSSV-infected P. japonicus, infection could be resolved only after three, 8 lg injections of vp28 siRNA (Xu et al., 2007). Wu et al. saw protective effects of siRNA for a variety of targets (see Table 1), but only when shrimp were subjected to virus challenge 24 h post-injection of siRNA. In P. monodon, siRNA against vp15 and vp28 genes did not provide signicantly more protection than the GFP control siRNA (Westenberg et al., 2005). A recent report from our lab further revealed that an optimized RNAi trigger for IMNV can be as short as 81 bp and provide 100% protection, but a shorter length (54 bp) sequence provided sub-optimal protection, providing some evidence for a threshold minimum length requirement for efcient induction of the antiviral state (Loy et al., 2012). This observation is in agreement with recent work that showed that only dsRNA of length greater than 50 bp, and not siRNAs, were capable of silencing native genes or stimulating transcription of RNAi pathway genes (Labreuche et al., 2010), and this points to a mechanistic explanation for limited successes for siRNAs as antivirals in penaeid shrimp.

3. Induced RNAi to mitigate shrimp disease: a vaccine approach? Whether or not an antiviral designed to mitigate disease in arthropods can be considered a vaccine is a point of some debate; this is because arthropod immune systems are understood to largely function in an innate capacity, thereby obviating the possibility of a memory response that involves antibody production. The concept of vaccination in shrimp in particular was reviewed by Johnson et al. (2008) with emphasis on examples of protective immunity conferred by protein-based vaccine approaches. The viral tolerance model (Flegel, 2007), and the possibility that provision or expression of specic viral surface proteins blocks virus receptors thereby preventing virus establishment and spread are potential mechanisms that underlie the observed protective immunity, but more specic mechanistic studies merit additional research (Johnson et al., 2008). If we take a more broad perspective of a vaccine as a molecular trigger that augments the immune response to protect the host in a pathogen-specic and long-term manner (spanning a signicant portion of the animals lifespan), then recent data from shrimp RNAi trigger development certainly support RNAi-based antivirals as aligning with the concept of vaccination. First, there is now indisputable evidence that RNAi is functioning as an immune response to virus infection in arthropod hosts, as reviewed recently for mosquitoes and Drosophila (Blair, 2011; van Mierlo et al., 2011), based on the following observations 1) SRRPs are functional during

virus infection and products of RNAi (in the form of small interfering RNAs) are evident in cell culture and host tissues during virus infection in mosquitoes and Drosophila 2) suppression of the RNAi pathway SRRPs results in widespread virus replication and lethal infection of otherwise non-cytolytic virus infections; for example, suppression of SRRPs in mosquito vector hosts, leads to widespread, high titer infections with Onyong nyong, Sindbis and Dengue virus (Keene et al., 2004; Campbell et al., 2008; SanchezVargas et al., 2009), and SRRP inhibition during Sindbis virus (SINV) infection results in high level mortality of mosquitoes (Myles et al., 2008; Cirimotich et al., 2009) as compared to SINV infection in the absence of RNAi inhibition. The same is true in Drosophila because SRRP null mutant ies have higher virus load and more pathogenic infections with Drosophila C and Drosophila X viruses, Cricket paralysis virus and Flock House virus (GalianaArnoux et al., 2006; van Rij et al., 2006; Wang et al., 2006; Zambon et al., 2006). Finally, for viruses that depend on the death of the host for transmission, the RNAi immune response clearly constitutes as a signicant selective pressure because several virus genomes encode suppressors of RNAi. For example, Cricket paralysis and Drosophila C virus encode suppressors that function by inhibiting Ago2, or by binding dsRNA to prevent Dicer2 processing, respectively (Nayak et al., 2010). Viral suppressors of RNAi encoded by the viruses of shrimp have yet to be identied, but there is evidence that WSSV infection suppresses the RNAi effect (Robalino et al., 2007), and speculation that IMNV encodes a protein that has a dsRNA binding domain and could be functioning in this manner (Poulos et al., 2006; Nibert, 2007). Speaking to the point that a vaccine should stimulate a pathogen specic response, the data summarized here show clearly that RNAi-based antivirals provide protection in a pathogen- and sequence-specic manner, such that some RNAi targets provide negligible protection, while others provide 100% protection from disease (see Table 1). For example, a dsRNA directed to a nonstructural protease from YHV provided 100% protection from disease in P. monodon at a dose of 25 lg/5 g animal (Yodmuang et al., 2006), and a dsRNA directed to ORF1/2 in PstDNV, which encodes non-structural protein NS1, provided complete virus clearance in L. vannamei as measured by PCR (Ho et al., 2011). In the case of WSSV and TSV, a small degree of disease protection was provided by injecting animals with a non-specic (to both host and virus) dsRNA sequence (see also Table 1). This was not the case with IMNV, for which no protection was observed in animals subjected to dsRNA for enhanced Green Fluorescent Protein (eGFP) (Loy et al., 2012). Furthermore, in several studies, non-specic dsRNA has been shown to provide this effect in a short-term manner; indeed, this observation led Robalino et al. to speculate that two pathways are activated in shrimp upon injection with dsRNA, an unspecied antiviral innate immune response, and an RNAi response (Robalino et al., 2007). In our experience, dsRNA for a non-specic sequence provides low-level and short term protection from WSSV (Fig. 1), but not for IMNV (Loy et al., 2012). Therefore, non-specic RNAi triggers do enhance shrimp antiviral immunity, but do not provide the protection of virus-specic RNAi triggers, and dont universally produce an antiviral effect. A potential stumbling point to dening RNAi triggers as vaccines for disease protection in shrimp is that RNAi capacity varies widely amongst arthropod species such that the long-term potential of the response comes into question. For example, the our beetle, Tenebrio molitor, has such a robust RNAi capacity, with functional gene suppression in virtually any cell type (Miller et al., 2008) and RNAi-based gene suppression being passed onto progeny. By contrast, in Drosophila, RNAi gene suppression is most often short-term and limited to a few tissue types (Miller et al., 2008). Because the lifespan of the cultured shrimp species represented in the literature is substantially longer than that of other

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Fig. 1. WSSV survival in an RNAi antiviral trial in L. vannamei. Shrimp at 35 g were injected with 5 lg VP19 dsRNA, a non-specic dsRNA for enhanced Green Fluorescent Protein (dseGFP), or with physiological saline. Three days later, animals were injected with puried WSSV. For each group, 30 shrimp were vaccinated and challenged in 3 replicate tanks (10/tank). Surviving animals were subjected to a second injection with WSSV at 25 days post vaccination.

well-studied arthropod species that are hosts to virus infection, there is perhaps a unique opportunity to study the long-term antiviral potential of RNAi. A key difference in the RNAi capacity is SID1 encoding genes that function as RNAi trigger receptors on cells and facilitate systemic spread of the effect. As noted above, SID-1 is present in the genome of penaeid shrimp and is functional during an RNAi event (Labreuche et al., 2010). There is evidence of systemic spread of antiviral RNAi in Drosophila through a nonSID 1 mechanism via genes involved in endocytosis, but the effect is short-lived such that high dose antiviral dsRNA suppression lasted only 5 days (Saleh et al., 2009). In Fig. 1, we show that dsRNA for VP19 (provided at a 5 lg dose to 35 g animals) provides high level and long-term protection from multiple challenges with WSSV, whereas a non-sequence specic dsRNA (eGFP) provided very high level protection upon rst exposure, but only 30% protection upon a second challenge. Mejia-Ruiz et al. showed that dsRNA for VP28 and VP26 provided 50% and 31% protection, respectively, at 30 days post-initial infection and repeated WSSV challenge every 10 days (Mejia-Ruiz et al., 2011). In the case of IMNV infection, vaccination with dsRNA provides 100% infection from disease and mortality upon challenge 2 and 10 days post-vaccination (dpv), and challenge at 2 dpv continues to provide that level of protection even 52 days later with a second challenge at a 100X higher dose (Loy et al., 2012). Seeing this level of protection at 10 and 52 dpv is an unprecedented result, given that in most studies the challenge is provided at 5 dpv or less. Therefore, pathogenand gene-specic RNAi triggers can provide long-term protection in shrimp from disease in support of the vaccine concept for RNAi in the host-pathogen systems that exist between shrimp and pathogenic viruses. 4. Developing the vaccine approach The data presented herein show that dsRNA-based antivirals provide signicant, sometimes long-term protection from a wide variety of highly pathogenic shrimp viruses. Given the economic importance of cultured shrimp and the devastating nancial toll of disease outbreaks, there is signicant need to further develop these antivirals as vaccines. Based on the data from viruses that represent single- or double-stranded RNA and DNA viruses, we conclude that there is no clear pattern to predict efcacy of RNAi trigger efcacy based on the virus gene product that is targeted. Indeed the RNAi triggers represented in Table 1 show that this approach can be effective at preventing disease by interfering with viral proteins that are produced in low or high abundance (with corresponding variation in transcript levels that have to be

suppressed) at the beginning or end of an infectious cycle. Therefore, the most effective target cannot necessarily be predicted according to protein function. In fact, our experience revealed an unexpected target, a protein cleavage product with unknown function, as the most effective target for RNAi in the IMNV genome (Loy et al., 2012). Furthermore, the protective effect was enhanced using one of ve different specic, signicantly truncated target regions within the coding sequence, as revealed using an iterative approach to trigger development (Loy et al., 2012). With highly effective antiviral RNAi triggers developed for many viruses of shrimp, the next obstacle to application of nucleic-based vaccines for cultured aquatic invertebrates is a practical, cost-effective delivery system. Although this seems a daunting challenge given the scale and environment in which these vaccines must be applied, there is ample reason to be optimistic that creative solutions are forthcoming. There is evidence to show that RNAi triggers are effective in vivo when delivered orally in a variety of arthropod species, including terrestrial pest and benecial insects (Hunter et al., 2010; Wuriyanghan et al., 2011; Zhu et al., 2011), aquatic (larval) mosquitoes (Zhang et al., 2010), and penaeid shrimp. Further, there are a number promising strategies emerging for oral delivery of RNAi triggers to cultured shrimp, including provision of dsRNA-producing bacteria or viral vectors in feed, as reviewed previously (Shekhar and Lu, 2009). Acknowledgments Support for some of the experimental data presented herein was provided by USDA SBIR WSSV Award 2010-33610-20936. The authors extend special thanks to Tasha Obrin (Iowa State University undergraduate program in Biological/Pre-Medical Illustration) for illustrating the graphical abstract. References
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