Evidence that the major hemolymph protein of the Pacific oyster, Crassostrea gigas, has antiviral activity against herpesviruses

Antiviral Research 110 (2014) 168–174

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Evidence that the major hemolymph protein of the Pacific oyster, Crassostrea gigas, has antiviral activity against herpesviruses Timothy J. Green a,⇑, Nick Robinson a,b, Tim Chataway c, Kirsten Benkendorff d, Wayne O’Connor e, Peter Speck a a

School of Biological Sciences and Australian Seafood Cooperative Research Centre, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia Nofima, P.O. Box 210, N-1431 Ås, Norway Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, SA, Australia d Marine Ecology Research Centre, Southern Cross University, P.O. Box 157, Lismore, NSW 2480, Australia e Industry & Investment NSW, Port Stephens Fisheries Institute, Taylors Beach, NSW 2316, Australia b c

a r t i c l e

i n f o

Article history: Received 24 June 2014 Revised 15 August 2014 Accepted 18 August 2014 Available online 27 August 2014 Keywords: Crassostrea Cavortin Extracellular superoxide dismutase Antiviral Herpesvirus

a b s t r a c t Viruses belonging to the family Malacoherpesviridae currently pose a serious threat to global production of the Pacific oyster, Crassostrea gigas. Hemolymph extracts from C. gigas are known to have potent antiviral activity. The compound(s) responsible for this broad-spectrum antiviral activity in oyster hemolymph have not been identified. The objective of this study was to identify these antiviral compound(s) and establish whether hemolymph antiviral activity is under genetic control in the Australian C. gigas population. Hemolymph antiviral activity of 18 family lines of C. gigas were assayed using a herpes simplex virus type 1 (HSV-1) and Vero cell plaque reduction assay. Differences in antiHSV-1 activity between the family lines were observed (p < 0.001) with heritability estimated to be low (h2 = 0.21). A glycoprotein that inhibits HSV-1 replication was identified by resolving oyster hemolymph by native-polyacrylamide gel electrophoresis (PAGE) and assaying extracted protein fractions using the HSV-1 and Vero cell plaque assay. Highest anti-HSV-1 activity corresponded with an N-linked glycoprotein with an estimated molecular mass of 21 kDa under non-reducing SDS–PAGE conditions. Amino acid sequencing by tandem mass spectrometry revealed this protein matched the major hemolymph protein, termed cavortin. Our results provide further evidence that cavortin is a multifunctional protein involved in immunity and that assays associated with its activity might be useful for markerassisted selection of disease resistant oysters. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Marine molluscs, such as oysters, have no clear evidence of adaptive immunity (Bachere et al., 2004), yet thrive in the oceans, which are rich in viruses (Bergh et al., 1989; Suttle, 2007). Despite the extraordinary abundance of viruses in the ocean, oysters live in high densities (i.e. oyster reefs), constantly filter the surrounding seawater for food and can live for several decades (Philipp and Abele, 2010). These observations suggest oysters have evolved an effective innate immune system to counteract viral infection. It is therefore not surprising that numerous studies have reported the presence of potent and broad-spectrum antiviral compounds in oyster tissue and hemolymph (Carriel-Gomes et al., 2006; Defer

⇑ Corresponding author. Current address: Department of Biological Sciences, Macquarie University, NSW 2109, Australia. E-mail address: [email protected] (T.J. Green). http://dx.doi.org/10.1016/j.antiviral.2014.08.010 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.

et al., 2009; Green et al., 2014; Olicard et al., 2005a,b; Zeng et al., 2008). The Pacific oyster, Crassostrea gigas, has been introduced from Asia to all other continents, except Antarctica (Hedgecock et al., 2005). It has formed the basis of a global aquaculture industry with one of the largest annual productions of any marine organism. The intentional movement of C. gigas, and other marine molluscs, around the world to develop aquaculture industries has aided the introduction of bivalve associated organisms, such as viruses, to new areas (Breener et al., 2014; Murray et al., 2012). Translocation of C. gigas outside its natural distribution range has also exposed it to new viruses (Breener et al., 2014; Murray et al., 2012). Not surprisingly, a new variant of the Ostreid herpesvirus type 1, termed OsHV-1 lvar, has emerged globally and has had a devastating impact on world production of C. gigas (Jenkins et al., 2013; Martenot et al., 2011; Segarra et al., 2010). There are very few management options that oyster farms can implement to reduce the

T.J. Green et al. / Antiviral Research 110 (2014) 168–174

detrimental impacts of disease. The only feasible options to counter the threat of viruses is to alter husbandry practices to reduce mortality (Paul-Pont et al., 2013), and develop disease resistant stock in the longer-term (Berthe et al., 2004). Oysters can be selectively bred for multiple production traits (Swan et al., 2007), including resistance to OsHV-1 lvar (Degremont, 2011, 2013). However, selective breeding is both costly and time-consuming (Davis and DeNise, 1998). New technologies, such as marker-assisted selection, can be applied to predict the genetic merit of individuals within a population for breeding and to enhance genetic gains (Davis and DeNise, 1998). The mollusc immune system is reliant on germ-line encoded antimicrobial effectors (Cooper, 2003) and it is known that the hemolymph of C. gigas contains an unidentified compound that interferes with herpesvirus replication (Olicard et al., 2005b). It seems plausible to select OsHV1 resistant oysters based on the activity of this antiviral compound. We undertook a study to identify this antiviral compound and determine whether hemolymph antiviral activity in C. gigas is under genetic control.

2. Materials and methods 2.1. Oysters, family lines and hemolymph sampling The oyster family lines used in this study were derived from single pair matings such that all individual were full siblings. The foundation parents of this population were obtained from three commercial hatcheries in Tasmania, together with some animals from feral Tasmanian populations, and were first spawned in the summer of 1997/98 (Swan et al., 2007). Successive selection and crosses of these family lines has since occurred with the emphasis primarily on weight, condition and shell characteristics. Adult oysters (2 years old) from eighteen families (spawned concurrently in 2009) were used in the current experiment. These families were held separately within the same nursery until approximately 3 mm in size. The oysters were then transferred to the field in Port Stephens, NSW, where all families were held in baskets on the same lease. Management practices were standardised for all families, with cleaning, grading and equipment changes all conducted concurrently. Periodically, oyster numbers within each family were reduced to maintain similar stocking densities. Hemolymph was withdrawn from the adductor muscle from 20 individuals per family using a 3-ml syringe equipped with a 19-gauge needle. Hemolymph samples were centrifuged at 1000g for 5 min before cell-free hemolymph was frozen at 20 °C. Hemolymph samples were transported on dry ice to Flinders University of South Australia using a commercial courier. Adult oysters were provided by Zippel Enterprises Pty Ltd (Smoky Bay, South Australia) for characterisation of the antiHSV-1 protein (see below). These oysters were housed in a marine aquarium (salinity 35 ppt, 17 °C) at Flinders University and hemolymph was collected from these oysters as required.

2.2. Cell culture, HSV-1 & plaque reduction assay African green monkey kidney (Vero) cells were grown in Dulbecco’s modified Eagle medium (DMEM, Sigma #D6429) supplemented with 10% newborn calf serum (Sigma #N4762) and 1% antibiotics (+10,000 IU penicillin ml1, +10,000 lg streptomycin ml1; Gibco #15140) at 37 °C in a humidified atmosphere of 5% CO2. A well-characterised strain, SC16 (Speck and Simmons, 1991), of wild-type HSV-1 was propagated in Vero cells and supernatants from infected cell cultures were clarified by centrifugation 1500g for 10 min and stored in aliquots at 80 °C. HSV-1 titre

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was calculated from plaque numbers according to the method of Reed and Muench (1938). Cytotoxicity assays were conducted prior to commencement of plaque reduction assays to determine the concentration range of C. gigas hemolymph that is non-toxic to Vero cells (George et al., 1996). Vero cells were seeded at a concentration of 3  105 cells per well in 24-well plate and cell culture medium was adjusted to contain C. gigas hemolymph at a concentration of 0%, 5%, 10%, 15% and 20% (v/v). Cells were grown for 48 h at 37 °C and cell viability was assessed using a trypan blue exclusion assay. Vero cells were detached with trypsin (5 mg ml1), stained with 4% trypan blue in PBS for 30 min and the percentage of viable cells estimated using a compound microscope and Neubauer counting chamber. We considered an acceptable level of cytotoxicity to be when C. gigas hemolymph caused less than 10% of cell death (CD10). Cell viability percentages of various C. gigas hemolymph concentrations (v/v) were determined by plotting a dose–response curve and the CD10 was calculated by regression analysis. A C. gigas hemolymph concentration of 13% (v/v) caused 10% Vero cell death. The antiviral activity of C. gigas hemolymph against HSV-1 was determined by a plaque reduction assay following the methods of Dang et al. (2011). Vero cell monolayers were obtained in a 24-well plate (CorningÒ #3524), and 45 ll (8%, v/v) of oyster hemolymph was added to the cells in duplicate. Cells and oyster hemolymph were incubated for 15 min on a bidirectional rotator (Thermo Scientific #4631) before monolayers were infected with 50 p.f.u of HSV-1. Cells and virus were incubated for 2 days at 37 °C before monolayers were fixed with 10% formaldehyde and stained with 2% toluidine blue. Plaques were counted using an inverted light microscope and antiviral activity of the hemolymph was expressed as the percentage reduction in plaque number compared to controls (DMEM). 2.3. Estimate of anti-HSV-1 heritability An animal model, that accommodated data from replicated HSV1 plaque reduction assays on the same individual, was applied to estimate genetic parameters and explore non-genetic influences on phenotype. A Markov chain Monte Carlo (MCMC) method using a multi-trait generalized linear mixed effect model (glmm) in a Bayesian estimation framework, with animal breeding value fitted as a random effect, was used for the analysis (R Package MCMCglmm, (Hadfield, 2010), http://www.cran.r-project.org). Our main interest was to determine whether hemolymph anti-HSV-1 activity was under genetic control and the following model was fitted:

P% ¼ mu þ trial þ animal þ ID where, P% was the percent protection measured by the plague reduction assay, animal and ID are random animal effects (ID was the same as animal factor, but was used by MCMCglmm to dissociate individual records from the pedigree and give an indication of between individual variance) and mu represented unknown random residual effects. Duplicated measurements of HSV-1 protection were treated as different trials (so trial was fitted as a fixed effect in the model). Models were run using 300,000 burn-in iterations, followed by 1,000,000 iterations and a thinning rate of 500. Estimates of additive genetic variance and residual variance were calculated from the modes of the posterior distribution and a Bayesian equivalent of 95% confidence intervals was obtained by calculating the values of estimates that bound 95% of the posterior distributions. Narrow sense heritability (h2), or the proportion of total phenotypic variation that is additive genetic in origin, was estimated under the animal model described above as VA/(VA + VE + Ve), where VA, VE and Ve were variance attributed to additive genetic, permanent environ-

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mental effects unconstrained by pedigree and residual error effects, respectively. Heritability was considered significant when the 95% credible interval of the posterior distribution did not span zero. 2.4. Identification of oyster anti-HSV-1 compound Hemolymph proteins from 3 adult oysters (pooled) were separated using native-polyacrylamide gel electrophoresis (BIO-RAD, Mini-PROTEANÒ II Cell). Discontinuous gels, 0.75 mm thick, were prepared using native (no sodium dodecyl sulfate, no reducing agent) Tris–glycine buffered gel with the resolving gels (10% acrylamide/bis) prepared with 1.5 M Tris–HCl, pH 8.8, and the stacking gel (4% acrylamide/bis) prepared with 0.5 M Tris–HCl, pH 6.8. An aliquot of oyster hemolymph (0.3 ml) was resolved at 120 V for 2 h. The resolving gel was then divided into five horizontal strips and hemolymph compounds eluted overnight in 1 ml of 0.01 M phosphate buffered saline (PBS). Hemolymph fractions (20% v/v) were then assayed for antiviral activity using the HSV-1 and Vero cell plaque reduction assay described above. Estimation of molecular mass of protein bands in each hemolymph fraction was determined by electrophoresis on 14% SDS–PAGE gels alongside unstained Precision Plus Protein™ Standard (BIO-RAD #161-0363). SDS–PAGE gels were stained with SYPROÒ Ruby protein gel stain (Invitrogen #S12000), PierceÒ Silver Stain Kit (Thermo Scientific #24612) and periodic acid-Schiff (PAS) stain to detect glycosylated proteins. Glycosylation analysis was performed on the protein fraction that corresponded with highest anti-HSV-1 activity. N-deglycosylation was done under reducing conditions using peptide N-glycosidase F (PNGase F) and the GlycoProfile™ II Enzymatic In-Solution N-Deglycosylation Kit (Sigma #PP0201) with RNase B as a control. O-deglycosylation was done under reducing conditions using neuraminidase (New England Biolabs #P0720S) and O-glycosidase (New England Biolabs #P0733S) according to the manufacturers protocol with fetuin (New England Biolabs #P6042) as a control. The protein fraction that corresponded with highest anti-HSV-1 activity was subjected to digestion by addition of 200 ng of trypsin (100 ng/ll in 10 mM NH4HCO3 buffer, pH 7.8). After overnight digestion at 37 °C, the sample was analysed with a Thermo Orbitrap XL mass spectrometer fitted with a nanospray source (Thermo Electron, San Jos, CA). The samples were applied to a 300 mm id  5 mm C18 PepMap 100 precolumn (Dionex, Sunnyvale, CA) and separated on a 75 mm  150 mm C18 5 lm 100 Å column (Nikkyo Technos, Tokyo, Japan), using a Dionex Ultimate 3000 HPLC (Dionex) with a 55-min gradient from 2% ACN to 45% ACN containing 0.1% formic acid at a flow rate of 200 nl/min followed by a step to 77% CAN for 9 min. The mass spectrometer was operated in positive ion mode with one FTMS scan of m/z 300–2000 at 60,000 resolution followed by ITMS product ion scans of the six most intense ions with dynamic exclusion of 30 s with 10 ppm low and high mass width relative to the reference mass, an exclusion list of 500 and CID energy of 35%. Only multiply charged ions were selected for MS/MS. The spectra were searched with Thermo Proteome Discoverer version 1.2 (Thermo Electron) using the SEQUEST algorithm against a fasta file of the translated oyster genome (Zhang et al., 2012).

(1000g for 5 min), cavortin-free hemolymph (286,000g for 3 h) and purified cavortin using the HSV-1 and Vero cell plaque reduction assay described above. Fivefold dilutions of purified cavortin were prepared using 0.2 lm filtered-seawater and tested in the plaque reduction assay to determine if the antiviral activity is titratable. The mode of antiviral activity that cavortin exerts (i.e. virucidal or prevents virus infection/replication) was assessed by varying the addition and incubation times of cavortin relative to HSV-1/Vero cell incubation, similar to the previously described procedures (Dang et al., 2011; Olicard et al., 2005b). Briefly, purified cavortin was included in separate plaque reduction assays for each of the following times: (i) pre-incubated with Vero cells for 2 h prior to addition of HSV-1; (ii) added simultaneously with HSV-1; (iii) added 2 h after HSV-1 infection of Vero cells; and (iv) pre-incubated with HSV-1 at room temperature for 60 min prior to addition to Vero cells. 3. Results 3.1. Anti-HSV-1 of C. gigas family lines and heritability Anti-HSV-1 activity of oyster hemolymph was variable between family lines of C. gigas (Fig. 1A, ANOVA p < 0.001). The narrowsense heritability of this trait (Percent Protection) was calculated by MCMC-glmm and found to be significantly different from zero (h2 = 0.21 with 95% confidence intervals at 0.11 and 0.43 for 307 animals tested in 18 full-sibling families). 3.2. Identification and characterization of hemolymph anti-HSV-1 compound Anti-HSV-1 activity was observed in all protein fractions resolved by native-PAGE (Fig. 2B). The negative control (nativepolyacrylamide gel soaked in PBS) also displayed antiviral activity against HSV-1, but was not cytotoxic to the Vero cells. Highest anti-HSV-1 activity was observed in fraction 2 (Fig. 2B), which had similar anti-HSV-1 activity to the untreated hemolymph positive control. SDS–PAGE revealed fraction 2 contained a single band with an estimated molecular mass of 21 kDa under non-reducing conditions (Fig. 2A). However, following reduction, this band migrated with an estimated molecular mass of 30 kDa. This 21 kDa band was positively stained by SYPRO Ruby, silver and PAS stain, which is suggestive of a glycoprotein. Deglycosylation of the 21 kDa protein band with PNGase F, but not O-glycosidase, confirmed this finding with a protein migrating faster after SDS– PAGE under reducing conditions (Fig. 3). The antiviral activity of

2.5. Characterisation of oyster hemolymph antiviral protein A putative antiviral protein, termed cavortin, was identified in Section 2.4 (see Results). Cavortin was pelleted from oyster hemolymph by ultracentrifugation (286,000g for 3 h in a Beckman NTV90 rotor) according to Scotti et al. (2007) and the pellet resuspended in 0.2 lm filtered seawater. Purity of cavortin was assessed using SDS–PAGE in 14% acrylamide/bis gel and antiviral activity confirmed by assaying whole hemolymph, cell-free hemolymph

Fig. 1. Antiviral activity in the hemolymph (8% v/v) of selected Australian family breeding lines of Crassostrea gigas against herpes simplex virus type 1 (HSV-1). The antiviral activity of each breeding line was assessed on individual oysters. Cont. = HSV-1 + DMEM.

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Fig. 2. Identification of anti-HSV-1 protein in Crassostrea gigas hemolymph. (A) Hemolymph was fractioned based on native molecular weight and proteins in each fraction was visualised on a 14% non-reducing SDS–polyacrylamide gel followed by silver staining. (B) Each protein fraction was then assessed for anti-HSV-1 activity using a Vero cell plaque assay. Highest anti-HSV-1 activity corresponded to F2. M = protein marker; CFH = cell free hemolymph. Note: Concentration of CFH and protein fraction is 20% v/v. F1 is a treatment control from native-PAGE gel slab soaked overnight in PBS.

Fig. 3. Crassostrea gigas anti-HSV-1 protein subjected to N-linked (A) and O-linked (B) deglycosylation reactions. N-deglycosylation and O-deglycosylation was performed using reducing conditions and hence, cavortin migrates as a 30 kDa band. Oyster anti-HSV-1 is a N-linked glycoprotein. Electrophoresis was done in 14% polyacrylamide gels followed by silver staining. Lane M = biorad marker; 1 = RNase B (control); 2 = RNase B + PNGase F; 3 = oyster protein; 4 = oyster protein + PNGase F; 5 = fetuin (control); 6 = fetuin + O-glycosidase; 7 = oyster protein; 8 = oyster protein + O-glyosidase.

each hemolymph fraction was positively correlated to the abundance of this 21 kDa glycoprotein (Table 1, r = 0.89, p < 0.05). Antiviral activity did not correlate with any other band identified by SDS–PAGE (Table 1). LC/MS/MS analysis of this 21 kDa protein band revealed two peptides that matched the translated sequence of cavortin (Genbank #AY551094) with high confidence (Fig. 4). Sedimentation of CFH proteins, including cavortin, was performed by ultracentrifugation (Fig. 5A) and this hemolymph fraction did not have antiHSV-1 activity (<3% reduction in plaques, Fig. 5B). CFH proteins resuspended in sterile seawater had similar anti-HSV-1 activity to the acellular hemolymph fraction. This crude purification of cavortin was used to assess its mode of antiviral action.

Table 1 Antiviral activity of Crassostrea gigas hemolymph proteins is positively correlated with a 21 kDa protein (p < 0.05). Protein (kDa)

Pearson r

p (two-tailed)

21 55 75 142 250

0.8933 0.3777 0.0951 0.2663 0.5574

0.0411 0.5308 0.8791 0.6649 0.3289

3.3. Mode of antiviral action Fivefold dilutions of purified cavortin revealed its antiviral activity is positively correlated with its concentration (Fig. 6, R2 = 0.895, p < 0.05). Cavortin exerts its antiviral activity after HSV-1 infection of Vero cells because pre-incubation of cavortin with Vero cells for 2 h prior to HSV-1 infection or the addition of cavortin at the time of infection did not reduce the number of plaques compared to addition of cavortin at 2 h after HSV-1 was added to the Vero cells (ANOVA, p > 0.05). Likewise, cavortin did not have a virucidal effect (<1% reduction in plaque numbers when HSV-1 was incubated with cavortin prior to infecting the Vero cells, p > 0.05).

Fig. 4. Translated cDNA sequence of cavortin (Genbank #AY511094). Amino acid residuals highlighted in black are matches. Cysteines are boxed.

4. Discussion This study indicates that cavortin, the major hemolymph protein of C. gigas, has antiviral activity against herpesviruses and oysters can be selectively bred for increased hemolymph antiviral

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Fig. 5. Ultracentrifugation of Crassostrea gigas hemolymph to purify cavortin. (A) SDS–PAGE analysis (non-reducing) of hemolymph (F1a), cell-free hemolymph (F2a), ultracentrifuged hemolymph (F3a) and hemolymph proteins resuspended in seawater. (B) Hemolymph antiviral activity could be eliminated from hemolymph centrifuged at 286,000g (F3a) and restored when hemolymph proteins were resuspended in seawater (F4a).

Fig. 6. Fivefold dilutions of purified cavortin revealed its antiviral activity is positively correlated with its concentration (R2 = 0.895, p < 0.05). Cavortin was resuspended and diluted in 0.2 lm filtered-seawater. Cont. = HSV-1 + DMEM.

activity. We assessed hemolymph antiviral activity using a heterologous model because cell lines from marine molluscs do not currently exist (Bickham and Bayne, 2013) and OsHV-1 cannot be cultured in primary cell cultures from bivalves (Garcia et al., 2011; Olicard et al., 2005a). We chose to evaluate C. gigas antiviral activity in a mammalian fibroblastic cell line (Vero cells) infected with HSV-1 because this model has been successfully employed in other studies of molluscan antiviral activity (Dang et al., 2011; Green et al., 2014; Olicard et al., 2005a,b). These previous studies identified antiviral activity in C. gigas hemolymph (Olicard et al., 2005a,b; Zeng et al., 2008), but were unable to identify the compound responsible for antiviral activity. Research suggests a genetic component exists for OsHV-1 resistance, and that genetic selection could improve this trait (Degremont, 2011, 2013). In France, genetic gains were easily obtained by breeding from oysters that had survived field exposure to OsHV-1. Genetic selection will be more complicated in Oceania because oyster hatcheries that provide spat to industry are located outside of the estuaries where OsHV-1 occurs (Jenkins et al., 2013). The risk of horizontal or vertical transmission of OsHV-1 in the hatchery is real (Arzul et al., 2001, 2002; Barbosa-Solomieu et al., 2005) and could potentially aid the escape of OsHV-1 into uninfected locations. Therefore, hatcheries in the Oceania region require an alternative method to select progenitors with increased antiviral resistance. We chose to investigate whether hemolymph antiviral activity against HSV-1 could be utilised as an indirect marker of viral resistance. Our results provide evidence that the hemolymph anti-HSV-1 activity is variable between pair-mated families of C. gigas (Fig. 1A) and that this trait is heritable (h2 = 0.21). Narrow sense heritability theoretically ranges from one (variation is 100% explained by additive genetic effects) to zero (no additive genetic component). It appears our heritability estimate of hemolymph

antiviral activity (h2 = 0.21) is low compared to other C. gigas traits, such as shell pigmentation (h2 = 0.50) (Evans et al., 2009), body weight (h2 = 0.313) (Evans and Langdon, 2006a) and survival (0.36–0.71) (Evans and Langdon, 2006b). Our study also provides preliminary data to suggest that the antiviral activity of oyster hemolymph corresponds to a 21 kDa N-linked glycoprotein, termed cavortin. Cavortin was identified by fractionating the hemolymph using native-PAGE and assaying each fraction using the HSV-1 and Vero cell plaque reduction assay. Anti-HSV-1 activity in each fraction was positively correlated to the abundance of cavortin in the sample (Table 1). Antiviral activity of cavortin was confirmed using a second approach of sedimenting cavortin by ultracentrifugation according to Scotti et al. (2007) and assaying crude cavortin (F4b) and cavortin-free hemolymph (F3b) in the HSV-1 and Vero cell plaque reduction assay (Fig. 5). Cavortin-free hemolymph did not have anti-HSV-1 activity, whereas sedimented cavortin resuspended in sterile seawater retained a similar level of anti-HSV-1 activity as cell-free hemolymph (Fig. 5). Additional assays were carried out to determine whether cavortin affected HSV-1 infection or replication, and whether it had a direct virucidal effect. Our results suggest cavortin exerts its antiviral action after HSV-1 infection, perhaps by interfering with HSV-1 replication. This observation is supported by previous research demonstrating the hemolymph of C. gigas contains an unidentified compound that interferes with HSV-1 replication (Olicard et al., 2005b). We were unable to ascertain the mechanism to explain why different C. gigas family lines have different hemolymph anti-HSV-1 activities. It is feasible that family lines with high anti-HSV-1 activities have a higher concentration of hemolymph cavortin because we demonstrated that the antiviral activity of cavortin is titratable (Fig. 6). Previous research has demonstrated that the hemolymph concentration of Saccostrea glomerata EcSOD, the orthologue of C. gigas cavortin, is elevated in oysters selected for disease resistance (Green et al., 2009; Simonian et al., 2009). Cavortin was first characterised as an immune protein, which comprises of a single Cu/Zn superoxide dismutase domain and is capable of binding to lipopolysaccharide via a DDED motif (Gonzalez et al., 2005). The function of cavortin has subsequently been disputed within the literature with several studies finding no compelling evidence to suggest cavortin has superoxide dismutase activity (Green et al., 2009; Itoh et al., 2011; Scotti et al., 2007). It has been suggested the superoxide dismutase domain had been altered during the course of evolution resulting in a protein that primarily functions as a metal ion chaperone (Scotti et al., 2007). Regardless of its primary function, cavortin must have an important immune role because it is regularly identified to be differentially expressed in oyster family lines selected for increased resistance to parasitic and viral diseases (Green et al., 2009;

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Huvet et al., 2004; Normand et al., 2014; Simonian et al., 2009; Taris et al., 2009). Indeed, Normand and colleagues (2014) recently concluded that resistance or susceptibility of C. gigas to OsHV-1 maybe partly explained by mRNA expression levels of cavortin. The major hemolymph proteins of other marine invertebrates also have multiple functions, including those involved in immunity (Coates and Nairn, 2014; Destoumieux-Garzon et al., 2001). Proteolysis of hemocyanin, the respiratory pigment of marine shrimp, produces polypeptides with potent antifungal activity (Destoumieux-Garzon et al., 2001). Experimental infections of shrimp result in elevated levels of hemocyanin-derived antimicrobial peptides constituting an important innate immune response to infection (Destoumieux-Garzon et al., 2001). Hemocyanins isolated from other arthropods have antiviral activity against a wide range of viruses that impact aquaculture (reviewed by Coates and Nairn, 2014) and hemocyanin purified from the marine gastropod (Haliotis laevigata) has antiviral activity against HSV-1 (Zanjani et al., 2014). SDS–PAGE analysis of cavortin revealed its relative mobility decreased under reducing conditions and migrates as a 30 kDa band. This change in molecular weight is characteristic of bivalve EcSOD proteins (Green et al., 2009; Itoh et al., 2011). It has previously been suggested that cavortin is a monomer with intramolecular disulfide bonds (Gonzalez et al., 2005). Cysteine is the only amino acid group capable of forming disulfide bonds (Wedemeyer et al., 2000) and the amino acid sequence of cavortin (Genbank #AY511094) reveals it contains only four cysteines (Fig. 4). It is therefore plausible that cavortin forms intermolecular disulfide bonds with another peptide to produce a multiprotein complex and the antiviral activity may be attributable to this accessory peptide. In conclusion, we provide evidence that the antiviral activity of oyster hemolymph is a trait under genetic control and this antiviral activity corresponds to the major hemolymph protein, termed cavortin. Future research should ascertain whether the difference in hemolymph antiviral activity between oyster families results from varying hemolymph concentrations of cavortin or different isoforms of cavortin. Further functional analysis of cavortin may lead to a reliable method to indirectly select C. gigas resistant to OsHV-1. Acknowledgements The authors acknowledge the financial assistance provided by the Australian Seafood Cooperative Research Centre (Project No. 2001/758). The authors would also like to acknowledge ASI Pty Ltd (Matt Cunningham) and Zippel Enterprises Pty Ltd (Gary Zippel) for the donation of oyster samples and their constructive advice. References Arzul, I., Nicolas, J.-L., Davison, A.J., Renault, T., 2001. French scallops: a new host for ostreid herpesvirus-1. Virol. J. 290, 342–349. Arzul, I., Renault, T., Thebault, A., Gerard, A., 2002. Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults. Virus Res. 84, 151– 160. Bachere, E., Gueguen, Y., Gonzalez, M., de Lorgeril, J., Garnier, J., Romestand, B., 2004. Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas. Immunol. Rev. 198, 147–168. Barbosa-Solomieu, V., Degremont, L., Vazquez-Juarez, R., Ascencio-Valle, F., Boudry, P., Renault, T., 2005. Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas). Virus Res. 107, 47– 56. Bergh, O., Borsheim, K.Y., Bratbak, G., Heldal, M., 1989. High abundance of viruses found in aquatic environments. Nature 340, 467–468. Berthe, F.C.J., Roux, F.L., Adlard, R.D., Figueras, A., 2004. Marteiliosis in molluscs: a review. Aquat. Living Resour. 17, 433–448. Bickham, U., Bayne, C.J., 2013. Molluscan cells in culture: primary cell cultures and cell lines. Can. J. Zool. 91, 391–404.

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