Gill-associated virus and recombinant protein vaccination in Penaeus monodon

Darren J. Underwood a, Jeff. A. Cowley b, Melony J. Sellars c, Andrew C. Barnes a,d,Marielle C.W. van Hulten a,1, Karyn N. Johnson a,!a School of Biological Sciences, The University of Queensland, St. Lucia, Qld 4072, Australiab CSIRO Food Futures National Research Flagship, CSIRO Livestock Industries, Queensland Biosciences Precinct, St. Lucia, Qld 4067, Australiac CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric Research, Cleveland, Qld 4163, Australiad Centre for Marine Studies, The University of Queensland, St. Lucia, Qld 4072, Australia

a b s t r a c ta r t i c l e i n f o

Article history:Received 6 July 2010Received in revised form 26 August 2010Accepted 26 August 2010

Keywords:Gill-associated virusBlack Tiger Shrimp (Penaeus monodon)Invertebrate immunityEnvelope glycoproteinsNucleocapsid

Recent studies in penaeid shrimp and various crustaceans showing speci!c antiviral responses to the DNAbasedWhite spot syndrome virus (WSSV) induced by pre-exposure to viral proteins are of particular researchinterest as invertebrates lack the classical adaptive immune response characterised in vertebrate animals.However, this protein-induced defence response has not been reported for any other invertebrate virus, andthemechanisms remain unknown. Herewe investigated the generality of this defence response by challengingPenaeusmonodon (Black Tiger shrimp)with the RNAbasedGill-associated virus (GAV) following pre-exposureto fragments of theGAVgp116 envelope glycoprotein or to the p20 nucleocapsid protein. Shrimpwere injectedtwice with recombinant GAV protein fragments expressed and puri!ed from bacteria before challenge withGAV. Protein vaccination did not protect the shrimp, with mortality rates in shrimp injected with any of theGAV proteins not differing from shrimp either not injectedwith protein or injectedwith a non-speci!c protein.In addition, there was no signi!cant difference in GAV RNA loads amongst the virus challenged shrimp groups.The data suggest that the ef!cacy of protection following protein-based vaccination may vary amongstdifferent shrimp viruses.

Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction

Viral infections occur commonly in both wild and cultured shrimp,potentially resulting in morbidity affecting growth and retail quality,and in some cases can be responsible for mass mortalities causingmajor production losses (Flegel, 1997). Globally, one of the mostdevastating viral pathogens in shrimp culture is White spot syndromevirus (WSSV) (Lightner, 1996; Wang et al., 2000) and in Australia,Gill-associated virus (GAV) has been linked to morbidity andmortalities in farmed Penaeus monodon (Spann et al., 2000; Cowleyet al., 2002; Callinan and Jiang, 2003). Despite the substantial impactof viral diseases on shrimp aquaculture, there are critical gaps in ourunderstanding of crustacean antiviral response mechanisms (Liuet al., 2009).

Penaeid antiviral responses mediated by genes such as P. monodonantiviral (PmAV) (Luo et al., 2003), anti-lipopolysaccharide factor (Liu

et al., 2006; Robalino et al., 2007; de la Vega et al., 2008) and C-typelectins (Liu et al., 2007; Ma et al., 2007; Zhao et al., 2009) have beenidenti!ed and, in some cases, undergone functional analysis. Togetherwith these inducible defence responses, shrimp can also mount adsRNA-based RNA silencing response (Hannon, 2002; Ding andVoinnet, 2007). Despite the expanding knowledge of the defencemechanisms that crustaceans can invoke to protect themselves againstviral infection, detail on how these mechanisms function remainsunknown.

The invertebrate defence against pathogens includes someinducible responses (Hoffmann, 2003; Kurtz, 2005; Kurtz andArmitage, 2006). There is growing evidence that invertebrates,including crustaceans, possess some form of ‘immune memory’ or‘priming’ mechanism that can be stimulated by past exposure to apathogen (Carius et al., 2001; Kurtz and Franz, 2003; Little andKraaijeveld, 2004; Pham et al., 2007; Schneider, 2007). In the case ofWSSV, protection can be induced by sub-lethal infection or exposureto inactivated WSSV or recombinant viral protein components (Wuet al., 2002; Namikoshi et al., 2004; Witteveldt et al., 2004a,b; Routet al., 2007). For example, Penaeus japonicus infected with a sub-lethal dose of WSSV have shown greater survival to subsequentchallenge with a lethal dose of the virus (Wu et al., 2002). Shrimpinjected with recombinant WSSV envelope proteins VP26 and VP28,

Aquaculture 308 (2010) 82–88

! Corresponding author. Tel.: +61 7 3365 1358; fax: +61 7 3365 1655.E-mail address: karynj@uq.edu.au (K.N. Johnson).

1 Current address: Intervet International B.V., Wim de Körverstraat 35, Boxmeer, TheNetherlands.

0044-8486/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2010.08.027

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either puri!ed from bacteria or synthesised in cells from eukaryoticexpression plasmids, can also be protected against developingdisease following challenge (van Hulten et al., 2000, 2001; Nami-koshi et al., 2004; Witteveldt et al., 2004b), as can shrimp fedrecombinant VP28 protein (Witteveldt et al., 2004a). Moreover,shrimp injected with plasmid DNA constructs expressing envelopeproteins VP28 and VP281 can be protected effectively whilst othersexpressing nucleocapsid proteins VP15 and VP35 remained suscep-tible to disease (Rout et al., 2007).

Whilst this growing evidence suggests that protein-based ‘vac-cines’ might have general applicability in crustaceans, protection hasnot been reported for viruses other than WSSV. Determining whethershrimp can be protected similarly against other viruses may provideinsight into the mechanism of protection. Of the viruses that causemortalities in shrimp species farmed in north-eastern Australia(Owens and Hall-Mendelin, 1990; Spann and Lester, 1997), GAV hasbeen examined for several reasons, the foremost of which being itsprevalence in wild and farmed P. monodon. GAV also has the ability tocause a disease and mortalities similar to that caused by the morehighly virulent Yellow-head virus (YHV) that continues to causeproduction losses in shrimp farmed in South-east Asia.

GAV generally presents as an asymptomatic infection, but wheninfection loads increase to high levels due to stress and other factors,acute infection leading to morbidity and death can occur (Spann et al.,1997; Cowley et al., 2004a; de la Vega et al., 2004). Unlike WSSV,which has a ~300 kbp dsDNA genome and is classi!ed as the typespecies of the Whispovirus genus of the Nimaviridae, GAV possesses a26.2 kb ssRNA genome and is the type species of the Okavirus genus inthe Roniviridae. In addition to containing a much smaller RNAgenome, virions of GAV and YHV are far less complicated than those ofWSSV comprising only a nucleocapsid protein (p20) and twoglycoproteins (gp116 and gp64) that protrude from the virion lipidenvelope (Cowley et al., 2000, 2004b; Cowley and Walker, 2002;Fauquet et al., 2005).

To investigate whether it might be possible to protect P. monodonagainst GAV injection with recombinant GAV protein componentsprior to challenge, selected regions of the gp116 envelope protein, andthe p20 proteinwere expressed as His6-fusion proteins in bacteria andpuri!ed for use as potential ‘immunogens’. The shrimp weresubsequently challenged with a minimum lethal dose of a GAV andassessed for protection.

2. Materials and methods

2.1. Expression and puri!cation of recombinant GAV proteins

Genome regions encoding portions of the predicted gp116ectodomain that protrudes from the virion envelope (Cowley andWalker, 2002; Jitrapakdee et al., 2003) were reverse transcribed,ampli!ed by PCR and cloned into pGEM-T (Promega). The sequenceof PCR primers used to amplify regions designated envelope-1(882 bp) and envelope-2 (1095 bp), residing toward the N- and C-terminal regions of gp116, respectively, is detailed in Table 1. Theconstructs pGEM-envelope-1 and pGEM-envelope-2 were se-quenced and the viral sequences were sub-cloned into pET28a(QIAGEN) for in-frame expression as His6-tagged fusion proteins.The PCR primer sequences used to amplify the GAV ORF2 geneencoding the p20 nucleocapsid protein and its cloning into pQE10(QIAGEN) and expression as a His6-tagged fusion protein are describedelsewhere (Cowley et al., 2004a).

Following plasmid DNA transformation into E. coli BL21 cells,bacterial colonies were selected on LB agar plates containingkanamycin. Several single colonies were cultured overnight and 1 mlwas used to inoculate 50 ml of LB containing kanamycin. Bacterialcultureswere agitated in conical "asks at 37 °C for ~2 h until the OD600

reached between 0.4 and 1.0. Isopropyl !-D-1-thiogalactopyranoside

(IPTG) was then added to a !nal concentration of 1 mM and agitationcontinued for approximately 2.5 h.

His6-fusion proteins in bacterial lysates prepared by sonication asper the TALON Metal Af!nity Resin kit (Clontech) protocol werevisualised in SDS-PAGE gels stained with Coomassie Brilliant Blue(CBB) and by Western blotting using anti-His6 antibody labelledwith alkaline phosphatase (Sigma). His6-fusion proteins werepuri!ed from bacterial lysates by gravity-"ow chromatographyusing Ni-agarose columns in the TALON Metal Af!nity Resin kit(Clontech) following the manufacturer's instructions. His6-fusionproteins that bound to the TALON resin were eluted in imidazoleelution (IE) buffer and the protein concentrations were determinedby Bradford assay using standards prepared from serially dilutedbovine serum albumin (BSA) (BioRad). SDS-PAGE followed by CBBstaining was used to assay protein purities.

2.2. Shrimp culture

Post-larval stage 170 P. monodon weighing 3–4 g were collectedfrom farm ponds by cast netting. Shrimp were transported, weighedand stocked in circular 100 l experimental tanks within 2 h of capture.Experimental tanks received "ow through natural seawater at a rateof 0.6 l/min, which maintained the temperature at 28±2 °C. Eachtank had a total volume of 80 l with light aeration and an opaquewhite lid to reduce light intensity. Replicates of three or four for eachof the seven treatments were distributed randomly around thefacility. Each tank contained !ve shrimp weighing between 3 and3.5 g and !ve shrimp weighing between 3.5 and 4 g (10 shrimp pertank). Commercial P. japonicus pellet mix (Lucky Star) was fed adlibitum twice a day and waste was removed by siphoning every 2–3 days. The number of shrimp alive in each tank was recorded early inthe morning and late in the afternoon, and any dead shrimp observedwere recorded and removed from tanks.

2.3. Shrimp protein injection and GAV challenge

A GAV inoculum was produced from the soft head tissues ofP. monodon that had become moribund following experimentalinjection of a similar tissue extract derived from P. monodon withhigh-level acute GAV infections. The soft tissue was diluted in sixvolumes of 0.22 "m !lter-sterilised shrimp salt solution (10 mMHEPES, 450 mM NaCl, 10 mM KCl, 10 mM EDTA pH 7.2–7.5) andhomogenised on ice until smooth using an Ultra-Turrax blender. Thehomogenate was centrifuged at 1125 g for 10 min at 4 °C and thesupernatant was further clari!ed by ultra-centrifugation at 40 572 gfor 20 min at 5 °C using an SW28 rotor. The supernatant was !lteredthrough a 0.45 "m !lter, snap frozen on dry ice and stored in 1.0 mlaliquots at !80 °C.

Table 1Primers for ampli!cation of GAV envelope glycoprotein regions.

Primer name Restrictionsite

Sequence (5"–3") Locationa

Envelope-1forward

BamHI GGATCCAAGGAGCCTGAAACATTAb 685–702

Envelope-1reverse

SalI GTCGACCTACGTGTTATATTCACAATCGb 1548–1566

Envelope-2forward

BamHI GGATCCATGAGCAACGCTAATAAGb 1567–1584

Envelope-2reverse

HindIII AAGCTTCTATGTATAAGCTGCAGAATCb 2644–2661

a Nucleotides of ORF3 that the primer sequence corresponds to.b Sequence in italics corresponds to restriction site; bolded sequence corresponds to

stop codons. The underlined sequences correspond to the nucleotides of ORF3.

83D.J. Underwood et al. / Aquaculture 308 (2010) 82–88

The GAV-infected shrimp tissue used to prepare the inoculum alsocontained high levels of Mourilyan virus (MoV) (Cowley et al., 2005).Thus to knock-down MoV replication following challenge, a double-stranded RNA (dsRNA) speci!c to a 723 nt portion of the MoV L RNAsegment was expressed in E. coli from the dual-T7 promoter plasmidpL4440, puri!ed using TRIzol reagent (Invitrogen), quanti!ed andstored at !80 °C.

Seven GAV bioassay treatments composed the trial, the negativecontrol P. monodon treatments were either (1) not injected or(2) injected with IE buffer and mock challenged with IE buffercontaining MoV dsRNA. The positive control treatment groups wereinjected with (3) IE buffer or (4) BSA in IE buffer as a non-speci!cprotein control, followed by injection of the diluted GAV inoculumcontaining MoV dsRNA. The test treatments were injected with therecombinant proteins (5) His6-p20, (6) His6-envelope-1 or (7) His6-envelope-2 before challenge with GAV inoculum containing MoVdsRNA as detailed in Table 2. Except for the two negative and thepositive control groups 1, 2 and 3, which included 3 replicate tanks,each experimental group included 4 replicate tanks. Protein wasdiluted to a concentration of 0.5 "g/"l and 40 "l was administered byintramuscular injection into the 3rd abdominal segment, resulting ina dose of at least 5 "g protein per gram of shrimp. Prior to thebioassay shrimp were vaccinated with each recombinant proteinwithout GAV challenge and mortality was analysed to ensure thatrecombinant proteins alone did not cause mortality (data notshown). A second booster dose of each protein was injected 4 daysafter the initial injection followed by the similar injection of 20 "lGAV inoculum 2 days later (Fig. 2A).

For MoV knock-down, 4 "g puri!ed MoV dsRNAwas injected intoeach shrimp together with the GAV and MoV inocula, resulting ina dose of approximately 1 "g dsRNA per gram. Based on otherdata, this dose was expected to be suf!cient to knock-down L RNAsegment levels and thus inhibit MoV replication (Yodmuang et al.,2006; Ongvarrasopone et al., 2008; Attasart et al., 2009).

2.4. Statistical analysis

To analyse differences in survival between groups, statisticalanalysis of survival was performed using the log-rank (Mantel–Cox)test with Graphpad Prism 5. Protection against GAV was calculated asrelative percent survival (RPS) [(1 ! treatment group mortality/positive control group mortality) ! 100] (Amend, 1981).

2.5. GAV infection load analysis

2.5.1. Shrimp samplingPleopods were sampled from shrimp non-destructively over the

course of the trial for analysis of GAV infection loads by RT-qPCR.Immediately following collection, pleopods were snap frozen on dry

ice and then stored at !80 °C until RNA extraction. Pleopods from 10untreated shrimp were sampled initially to determine the pre-existing baseline infection loads of GAV and MoV. Pleopods fromanother 10 shrimp injected with the GAV inoculum but that did notreceive protein were sampled at 4 days post-challenge to assay forincreases in GAV infection loads and whether MoV replicationhad been inhibited effectively by co-administration of MoV-speci!cdsRNA in the inoculum. Pleopods were also sampled from all shrimpin the trial at eight days post-challenge when mortality amongst thepositive control shrimp had reached approximately 40%.

2.5.2. RNA extraction and cDNA synthesisPleopods sampled from !ve shrimp from each treatment group

were each homogenised in 500 "l TRI-reagent (Molecular ResearchCentre) using 3 mm glass beads and agitation in a bead beater for atleast 1 min. Total RNA was extracted according to the manufacturer'sinstructions and quanti!ed using a NanoDrop spectrophotometer atOD260. Before cDNA synthesis, the RNA was treated with DNAse(Promega) to remove any traces of contaminating DNA. For cDNAsynthesis the pleopod RNA (500 ng) along with 333 pg !-luciferaseRNA (Promega), which was added to each reaction as an exogenousRNA control, was reverse transcribed using SuperScript III (Invitro-gen) and random hexamers (Promega) according to the manufac-turer's recommended protocol.

2.5.3. RT-qPCRRelative viral RNA levels were quanti!ed by RT-qPCR using the

Platinum SYBR green RT-qPCR supermix (Invitrogen) according to themanufacturer's protocol and a Rotor-Gene Q thermal cycler (QIAGEN)!tted with a 72 well rotor. A 2 "l aliquot of cDNA diluted 1:50 wasused in duplicate 10 "l reactions. The thermal cycling pro!le usedfor RT-qPCR was 95 °C for 2 min, 50 °C for 2 min, then 40 cycles of95 °C for 10 s, 60 °C for 10 s, 72 °C for 20 s followed by a 65 °C–95 °Cmelt analysis.

PCR primer sequences used for RT-qPCR are listed in Table 3.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elonga-tion factor-1 alpha (EF-1#) were selected for reference genes as theyhave been shown to be suitable for relative RT-qPCR analysis ofpenaeid immune genes (Dhar et al., 2009). PCR ef!ciency wascalculated for each primer pair used to detect the exogenousluciferase RNA control, GAPDH, EF-1#, GAV and MoV.

RT-qPCR data were analysed using the REST 2009 software(QIAGEN) in RG mode incorporating Cq take-off values from theRotor-gene software together with ampli!cation ef!ciencies deter-mined for each primer pair. Statistical analysis utilised both referencegenes using the REST 2009 software. The data were representedgraphically by comparing the change in GAV RNA load to the changein GAPDH RNA load for each sample set using a combination of qGeneand the REST 2009 software.

Table 2GAV bioassay.

Description Vaccination Challenge Shrimp perreplicate

(1) Negative control—no injection

None None 10

(2) Negative control Elution buffer MoV dsRNAa 10(3) Positive control Elution buffer GAV+20 "l MoV dsRNAa 10(4) Non-speci!c

protein controlBSAb GAV+20 "l MoV dsRNAa 10

(5) Nucleocapsid ORF2b GAV+20 "l MoV dsRNAa 10(6) Envelope-1 ORF3B-1b GAV+20 "l MoV dsRNAa 10(7) Envelope-2 ORF3B-2b GAV+20 "l MoV dsRNAa 10a 4 "g MoV dsRNA diluted in 0.3 M NaCl for a total injection volume of 40 "l.b Protein concentration of 5 "g/g of shrimp, diluted in elution buffer for a total

injection volume of 40 "l.

Table 3Primer pairs used for RT-qPCR analysis.

Target Sequences Target sequence

!-luciferase Forward: 5"-GGTGTTGGGCGCGTTATTTA-3" Control exogenousRNA (Promega)Reverse: 5"-CGGTAGGCTGCGAAATGC-3"

GAPDH Forward: 5"-GAGGTGGTGGCTGTGAATGA-3" GenBank accession:AI770197Reverse: 5"-GCCTTGACCTCCCCCTTGT-3"

EF-1#a Forward: 5"-TCGCCGAACTGCTGACCAAGA-3" GenBank accession:DQ021452Reverse: 5"-CCGGCTTCCAGTTCCTTACC-3"

GAV Forward: 5"-GGGATCCTAACATCGTCAACGT-3" GenBank accession:NC_010306Reverse: 5"-AGTAGTATGGATTACCCTGGTGCAT-3"

MoV Forward: 5"-TGTTACAAGCACACTGCATCTCA-3" GenBank accession:AY927991.1Reverse: 5"-GCTAGGGCAGACCACTTCACA-3"

a P. stylirostris primers (Dhar et al., 2002).

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3. Results

3.1. Expression and puri!cation of recombinant GAV protein components

The GAV p20 nucleocapsid protein and two segments of the GAVp116 envelope glycoprotein were expressed as His6-tagged recom-binant proteins in bacteria. SDS-PAGE and CBB staining of the His6-tagged fusion proteins present in bacterial cell lysates are shown inFig. 1. Compared to cell lysates of bacteria transformed with pQE10and pET28a (Fig. 1A), additional proteins of ~18 kDa, ~40 kDa and~45 kDa were detected in lysates of bacteria transformed withpQE10-p20, pET28a-envelope-1 and pET28a-envelope-2, respective-ly (Fig. 1B). Western blot analysis using an anti-His6 antibodydetected recombinant GAV proteins of the same size (Fig. 1C). ThepQE10-p20 protein and pET28a-envelope-2 protein migrated closeto their predicted masses of 18 kDa and 45 kDa. The pET28a-envelope-1 proteinmigrated to a position slightly above its predictedmass of 37 kDa. However we suggest that this is the correct proteinas it is not observed in the bacteria expressing empty vector, is His6-tagged and the clone was con!rmed to contain the correct DNAby sequencing. An additional unexpected band which may be adegredation product was observed in the Western blot analysis ofcell lysates of bacteria transformed with pET28a-envelope-2(Fig. 1C).

The His6-fusion proteins were puri!ed by af!nity chromatogra-phy using TALON Ni-columns and multiple protein samples wereprepared and puri!ed to generate suf!cient protein for the GAVbioassays. SDS-PAGE analysis showed each of the three, pooledpuri!ed His6-fusion proteins to be relatively pure (Fig. 1D).

3.2. GAV bioassay

In the positive control shrimp injected with GAV (treatment 3),cumulative mortality of 53% was reached at 11 days post-challengeand remained unaltered until the bioassay was terminated at 28 dayspost-challenge. At the end of the trial, relatively few shrimp had diedin themock-infected control groups 1 and 2 (2.5% and 3.3%mortality,respectively). Cumulative mortalities in the infected groups pre-injected with various proteins (groups 4—43%, 5—49%, 6—28% and7—52.5%) did not appear obviously different when compared to thepositive control group 3—47% (Fig. 2B). Relative percent survival(RPS) values calculated from the !nal shrimp mortality numbers ofgroups 4, 5, 6 and 7 compared to group 3 were !7.8% (P=0.95),3.8% (P=0.60), !34.6% (P=0.21) and 10.9% (P=0.52) respec-tively, and statistical analysis of the mortality curves using the log-rank test showed none to have signi!cant P values.

3.3. Viral load analysis

No increase in MoV RNA loads was detected in shrimp challengedwith the GAV inoculum containing the MoV L RNA segmentdsRNA. This suggests that the dsRNA had effectively inhibitedMoV replication (data not shown). GAV RNA loads increased in allgroups injected with GAV, and compared to the positive controlshrimp groups 3 and 4, none of the groups pre-exposed to thevarious GAV proteins (groups 5, 6 and 7) showed any statisticallysigni!cant (Pb0.05) decreases relative to GAPDH or EF-1# mRNAlevels (Fig. 3).

4. Discussion

Several studies with WSSV have shown that prior non-lethalexposure to the virus or to viral envelope proteins can induce aprotective response in penaeid shrimp (Caipang et al., 2008; Du et al.,2006; Namikoshi et al., 2004; Rout et al., 2007; Witteveldt et al.,2004a,b; Wu et al., 2002). Protection provided by recombinant

Fig. 1. SDS-PAGE analysis of His6-fusion proteins expressed in bacteria. (A) Proteins ofcell lysates of bacteria transformed with pQE10 (lane 1) or pET28a (lane 2) followingIPTG induction and analysis in a 12% SDS-PAGE gel stained with CBB. M = protein sizestandards (Fermentas). (B) IPTG-induced proteins expressed by bacteria transformedwith the plasmids pQE10-nucleocapsid (lane 1), pET28a-envelope-1 (lane 2) andpET28a-envelope-2 (lane 3). Proteins were resolved in a 10% SDS-PAGE gel and stainedwith CBBM=protein size standards (Fermentas). (C) IPTG-induced proteins expressedby bacteria transfected with the plasmids pQE10 (lane 1), pET28a (lane 2), pQE10-nucleocapsid (lane 3), pET28a-envelope-1 (lane 4) and pET28a-envelope-2 (lane 5).The pQE10 plasmids were transfected into M15 [pREP4] cells and the pET28a plasmidswere transfected into BL21 cells. Proteins were resolved in a 10% SDS-PAGE gel anddetected by Western blotting using an anti-His6 tag antibody. M= SeeBlue pre-stainedprotein size standards (Invitrogen). (D) Pools of proteins puri!ed by TALON Ni-af!nitychromatography and used in bioassays at a concentration of 0.25 "g/"l. Puri!ed proteinswere resolved in a 10% SDS-PAGE gel and stained with CBB. Puri!ed His6-p20nucleocapsid protein (lane 1), His6-envelope-1 protein (lane 2) and His6-envelope-2protein (lane 3). M = protein size standards (Fermentas).

85D.J. Underwood et al. / Aquaculture 308 (2010) 82–88

proteins has been relatively short term, lasting 10–30 days, butprotection lasting longer up to ~50 days has been recorded followingnon-lethal exposure to live virus or when eukaryotic expressionplasmids have been used for intracellular expression of recombinantproteins (Wu et al., 2002; Namikoshi et al., 2004; Witteveldt et al.,2004b; Rout et al., 2007). These data tend to suggest that persistentexposure to viral protein may be required for continued protection.Flegel (2007) has elaborated a model of viral accommodation thatsupports this view, in which virus infection sustained at low levels,whilst not rendering the host resistant to re-infection, can providesome speci!c memory response that can interfere with viruspathogenicity upon re-infection. Another model has been proposedin which cell surface receptors required for viral uptake might bindspeci!c viral proteins and thus block virus attachment and entry(Johnson et al., 2008). This model also accommodates why shrimpsusceptibility to infection and disease can re-emerge in the absence ofcontinued exposure to viral proteins. Without empirical data,however, the molecular mechanisms underpinning protein-basedprotection against WSSV infection and disease will remain a topic ofspeculation.

To investigate the potential for protecting shrimp against diseasecaused by a virus quite distinct from WSSV, recombinant proteinsexpressed in bacteria consisting of the p20 nucleocapsid protein andportions of the gp116 envelope glycoprotein of GAV were assessed inbioassays for their ability to protect against GAV induced mortality.Unlike WSSV however, in which shrimp could be protected by pre-exposure to WSSV envelope proteins (Wu et al., 2002; Namikoshiet al., 2004;Witteveldt et al., 2004a,b; Rout et al., 2007), pre-exposureto the GAV proteins provided no protection. Moreover, RT-qPCRquanti!cation of GAV RNA loads in the pleopods of P. monodon groupspre-exposed to any of the three GAV recombinant proteins showed nostatistical differences in virus load following challenge compared tothe positive control groups.

Despite the inability of the bacterially expressed recombinantGAV proteins to protect shrimp against GAV, it is possible that theprotective responses observed with WSSV might be generated

similarly with other shrimp virus types. For example, it is possiblethat the inability of the proteins to protect against GAV was due todifferences in the recombinant protein fragments compared to thatof the native gp116 glycoprotein. In infected shrimp cells nativegp116 protein, which is generated by the post-translational cleavageof the pp3 polyprotein translated from the ORF3 gene, is anchored toendoplasmic membranes and is processed through Golgi andendoplasmic membranes where it becomes highly decorated withcarbohydrate regions (Cowley and Walker, 2002; Jitrapakdee et al.,2003; Soowannayan et al., 2010). This is different from gp116 proteinexpressed in bacteria, where there is no capacity to process andpresent the protein similarly. The authentic structure and post-translational processing of the protein might impact on the responseelicited in animals. Consistent with this, in WSSV protection assays,an increase in protection was seen when eukaryotic recombinantproteins were used compared to bacterial proteins (Du et al., 2006).However in contrast to our results with GAV, recombinant bacterialenvelope proteins provided signi!cant protection against WSSV.It remains to be seen if GAV glycoproteins expressed in an inver-tebrate cell or yeast expression system might preserve theirstructural and functional integrity suf!ciently to afford protection.The lack of protection with GAV p20, which is a component of thenucleocapsid and thus not needed for cell attachment and/or entry, isconsistent with the results for WSSV nucleocapsid proteins (Routet al., 2007).

It is also possible that the nature of WSSV and GAV affects theirsusceptibility to protein-based protective responses. For example,WSSV contains a circular dsDNA genome and replicates in the cellnucleus, whilst GAV contains a positive-sense ssRNA genome andreplicates in the cell cytoplasm. Basic differences in their replicationprocesses might affect what protein-induced protective responsescan operate. Additional challenge trials with GAV or other penaeidviruses are needed to elucidate the mechanisms by which pre-exposure to viral protein can afford protection, as demonstratedfor WSSV using several systems of expressing and delivering viralprotein.

Fig. 2. Challenge model and accumulated survival data obtained in the GAV bioassays. (A) Protein administration and viral challenge time points. (B) Average percentage survivalof Penaeus monodon over 28 days following viral challenge of groups pre-exposed to various proteins. Standard error bar lengths were calculated from the replicate tanks foreach treatment.

86 D.J. Underwood et al. / Aquaculture 308 (2010) 82–88

Acknowledgements

The authors thank Min Rao and Erik DeVries for their technicalassistance as well as Stuart Arnold, NickWade, Greg Coman, EkaterinaNowak, Kate Bullen and Ian Underwood for their assistance withrunning the shrimp bioassays.

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