Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection*

Background: Melanization plays a major role in invertebrate defense. Results: Suppression of shrimp melanization increased the WSSV susceptibility. The viral protein, WSSV453, interferes the proPO system via PmPPAE2 inhibition. Conclusion: Shrimp melanization has an antiviral role. WSSV overcomes this by suppressing the host proPO proteinase cascade. Significance: The regulation of shrimp melanization during WSSV infection was first demonstrated. The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, sug-gesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2inhibiting activity of WSSV453.
White spot syndrome virus (WSSV), 3 an enveloped and double-stranded circular DNA virus, is one of the major shrimp pathogens that causes a cumulative mortality of up to 100% within a week and has consistently affected shrimp farming worldwide and led to drastic losses in shrimp production (1)(2)(3)(4). An insight into the molecular mechanisms underlying the shrimp-virus immune interactions is particularly essential for helping combat these viral infections and improving the cultured shrimp immunity.
In the shrimp Penaeus monodon, two proPO isoforms (PmproPO1 and PmproPO2) have been characterized, and the activation of zymogen PmproPOs into the active POs needs the proteolytic activity of the proPO-activating enzymes (PmPPAE) 1 and 2 (15,(22)(23)(24). The proPO system of P. monodon has been shown to play an important role in the defense against pathogenic bacterial and fungal infections (15,(22)(23)(24)(25), but no reports currently indicate a role in the antiviral immune response for the shrimp proPO system. However, the role of melanization in an antiviral response has been reported in some invertebrates. For example, in the mosquito Armigeres subalbalus, the proPO cascade is essential in the immune defense toward arboviruses, such as Semliki Forest virus, where inhibition of the melanization reaction leads to a higher level of virus replication and mosquito mortality (13). In the lepidopteran Manduca sexta, the reactive compound of melanization (5,6-dihydroxyindole) exhibits antibacterial, antifungal, and antiviral activities (18,19). In P. monodon, the previous finding has shown that melanization reaction products of shrimp exhibit antimicrobial properties toward the major bacterial and fungal pathogens (25). However, whether melanization in shrimp provides any defense against WSSV is unclear.
Based on previous studies in several shrimp, several genes, including the proPO-associated genes, have been identified as having an altered transcript expression level upon WSSV infection. For instance, in Litopenaeus vannamei and the crayfish Procambarus clarkii, the mRNA expression level of proPO genes was down-regulated after WSSV infection (26 -28). In Fenneropenaeus chinensis, it was found that FcPPAEIII, FcPPO1, and FcPPO2 were up-regulated, whereas FcPPAE1a and FcPPO3 were down-regulated in acute infections compared with that in latent infections (29). The change in these gene expression levels imply that the proPO system may play a role in response to WSSV infection. Moreover, in L. vannamei the level of PO activity significantly decreased at 48 h post injection (hpi) (30). However, the information on the mechanism of WSSV controlling or down-regulating the shrimp melanization reaction is still very limited and unclear.
The control and disabling of the host melanization was reported in M. sexta (31), where oviposition of the wasp Microplitis demolitor in M. sexta larvae uses the M. demolitor bracovirus (MdBV) that encodes for epidermal growth factor-like motif (Egf) proteins Egf1.0 and Egf1.5. These small serine proteinase inhibitors are capable of suppressing the melanization response of host insects by inhibiting the PPAEs and so inhibit the proteolytic cleaving of proPO into the active PO (32,33). These former reports lead to the question of whether proPO in P. monodon is controlled or suppressed upon WSSV infection.
In the present study, we investigated the role of the P. monodon proPO system in the immune response against WSSV infection. This study provides the first demonstration that melanization could have an antiviral role in shrimp and that as part of WSSV pathogenesis, the virus might have the ability to suppress the melanization response of the shrimp via the inhibition of the proPO proteinase cascade, analogous to that found in insects. Thus, this study reveals the crucial hostpathogen interaction during WSSV infection.

Shrimp and Virus Stock Preparation and Infection-Specific
pathogen-free black tiger shrimp (P. monodon) were obtained from the Shrimp Genetic Improvement Center (National Center for Genetic Engineering and Biotechnology, Surat Thani, Thailand). They were kept in laboratory tanks with diluted natural seawater (salinity of ϳ20 ppt) and reared for 7 days before use. The WSSV stock solution was prepared from the gills of WSSV-infected shrimp by homogenizing in PBS (pH 7.4) and then centrifuging at 400 ϫ g for 10 min at 4°C. The supernatant fluid was filtered through a 0.45-m pore size filter and used as the WSSV stock solution, stored in Ϫ80°C until used. The titer of the WSSV stock was determined by in vivo infection experiments. Shrimp were intramuscularly injected with the optimal dilution level of the WSSV stock solution in PBS at the lateral area of the fourth abdominal segment using a syringe with a 29-gauge needle. The mortality was recorded daily for 7 days, and the dose of the virus that caused 50% mortality (LD 50 ) within 3 days after injection was used for all subsequent viral infections of the shrimp.
Effect of WSSV Infection on proPO(s)-silenced Shrimp-The cumulative mortality of proPO(s)-silenced shrimp after WSSV infection was evaluated to examine the importance of the proPO system in the defense against WSSV. Double-stranded RNAs (dsRNAs) of PmproPO1 and PmproPO2 genes were generated in vitro using the T7 RiboMAX express large scale RNA production system (Promega) with the gene-specific primer pairs of T7PO1i-F/-R, PO1i-F/-R, T7PO2i-F/-R, and PO2i-F/-R (Table 1) as reported (22). Juvenile shrimp were intramuscularly injected with 25 l of 150 mM NaCl (sodium saline solution; SSS) containing either (i) PmproPO1 and PmproPO2 dsRNA (5 g each/g shrimp, wet body weight), (ii) GFP dsRNA (at the same concentration), or (iii) SSS only. At 24 hpi, a second 25-l injection as before but also containing WSSV (LD 50 dose) was administered. Thereafter, the number of dead shrimp was recorded daily over a 5-day period to ascertain the cumulative mortality. The experiments were performed in triplicate.
Effect of the PO-generated Reactive Compounds on WSSV Infectivity-The antiviral role of the reactive compounds produced by the shrimp proPO system was investigated in vitro by observing the survival rate of shrimp infected with in vitro melanization reaction-treated WSSV. The in vitro shrimp melanization reaction was prepared by incubating shrimp hemocyte lysate supernatant (HLS) (15 g/5 g shrimp), prepared as previously described (25), with Escherichia coli O127:B8 LPS (Sigma; L4130) at 0.5 g/5 g shrimp, laminarin (␤-1,3-glucan) from Laminaria digitata (Sigma; L9634) at 0.5 g/5 g shrimp, and dopamine (1 M) in 10 mM Tris-HCl (pH 8.0) for 15 min. Then the WSSV stock solution was treated with this melanization reaction at room temperature for 1 h. Thereafter, five groups of shrimp (5 g) were injected with (i) the melanization reaction-treated WSSV or, for the four control groups, with WSSV incubated with either (ii) HLS or (iii) dopamine and with (iv) dopamine ϩ HLS or (v) WSSV only. The number of surviving shrimp in each group was recorded daily over a 10-day period. The experiments were performed in triplicate.
The quantification of WSSV replication in shrimp after injection with the different solutions was examined by semiquantitative two-stage RT-PCR with the gene-specific VP28 primer pair ( Table 1). The hemolymph from three surviving shrimp in each group were collected at 5 days post injection (dpi), and the total RNA was extracted using an Illustra RNAspin mini kit (GE Healthcare). Then first strand cDNAs were reverse transcribed from the poly(A)-tailed mRNA using 180 g of DNA-free total RNA sample and 0.25 g of oligo(dT) 18 primer with the RevertAid first strand cDNA synthesis kit (Thermo Scientific), as per the manufacturer's protocol. The transcript expression level of the WSSV VP28 and host EF1-␣ genes were then evaluated using the cDNA and the specific VP28 and EF1-␣ primers ( Table 1). The PCRs were performed at 94°C for 1 min, followed by 25 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and 72°C for 5 min. The VP28 transcript levels were then normalized to that of the host EF1-␣ gene for each sample.
Determination of the PO Activity in the Hemolymph of WSSV-infected Shrimp-To examine the effect of WSSV infection on the shrimp proPO system, the PO activity of WSSVinfected shrimp at 1, 2, and 3 dpi were measured. Shrimp were divided into the two groups of the experimental (WSSV-infected shrimp) and the control (PBS-injected shrimp) groups. The shrimp hemolymph was withdrawn without using an anti-coagulant from the shrimp ventral sinus at 1, 2, and 3 dpi. The PO activity in the hemolymph, expressed as the amount of dopachrome formation from the L-3,4-dihydroxyphenylalanine substrate, was measured as previously reported (22). Briefly, total hemolymph proteins (2 mg of protein) in 435 l of Tris-HCl (10 mM, pH 8.0) were mixed with freshly prepared L-DOPA (3 mg/ml in water; Fluka). After incubation at room temperature for 30 min, 10% (v/v) acetic acid was added to the mixture, and the absorbance at 490 nm was monitored. The PO activity was recorded as ⌬A 490 /mg of total protein/min, and the protein concentration was determined by Bradford's method. In addition, the exogenous trypsin activation of the WSSV-infected and PBS-injected shrimp hemolymph was observed by adding trypsin (20 M) into both groups, and then the PO activity was determined as described above. All experiments were performed in triplicate and statistical analysis was performed using a one-way analysis of variance followed by Duncan's test.
Yeast Two-hybrid Assay-Yeast two-hybrid screens were performed based on the Clontech Matchmaker GAL4 two-hybrid system to identify which WSSV proteins potentially interact with PmPPAE2, a serine proteinase enzyme in the shrimp proPO system. To construct the PmPPAE2 bait vector, a mature peptide coding sequence of PmPPAE2 (GenBank TM accession number FJ620685) was amplified using Pfu DNA polymerase (Promega) with the specific primers PmPPAE2NcoI-F/PmPPAE2XhoI-R that contain 5Ј-flanking NcoI and XhoI restriction enzyme sites, respectively (Table 1). PCR amplifica-

Cloning and recombinant protein expression
tion was conducted at 94°C for 1 min, followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C and then a final 72°C for 10 min. Amplified products were digested with NcoI and XhoI restriction nucleases. The digested fragments were purified and ligated into the NcoI/SalI sites of pGBKT7 to yield the final PmPPAE2/pGBKT7 construct that was cloned and subsequently sequenced to ensure the correct and in-frame insertion.
For the yeast two-hybrid screening, the PmPPAE2/pGBKT7 construct obtained above was used as the bait to screen for interacting proteins (prey) from a WSSV DNA library fused with the AD domain of pGADT7 (34). Positive interactions were indicated by growth on the high stringency media lacking leucine, tryptophan, adenine, and histidine (SD/ϪL/ϪW/ϪA/ ϪH) and by a blue color change caused by 5-bromo-4-chloro-3-indolyl-␣-D-galactoside (X-␣-gal; Apollo Scientific) in the medium. Library plasmids from positive colonies were rescued in E. coli XL1-Blue cells and reconfirmed by yeast cotransformation. The positive prey plasmids were then subjected to DNA sequencing. The nucleotide sequences were compared with the deduced amino acid sequences against the GenBank TM database using the BLASTx program.
Cloning and Recombinant Protein Expression of PmPPAE2 and WSSV453-To investigate the biological role of the WSSV453 and PmPPAE2 proteins, recombinant (r)WSSV453 and rPmPPAE2 were expressed. First, the gene fragments encoding the respective serine proteinase (SP) domain of PmPPAE2 (SP-PmPPAE2) and the WSSV453 protein were amplified using Pfu DNA polymerase with the specific primer pairs SPPmPPAE2NcoI-F/PmPPAE2NotI-R and WSSV453PciI-F/ WSSV453NotI-R, respectively ( Table 1). The purified SP-PmPPAE2 and WSSV453 PCR products were digested with the corresponding restriction enzymes (Table 1) and ligated into the pET28b and pET32 expression vectors (Novagen), respectively. Then the respective recombinant plasmid was transformed into competent E. coli JM109, and positive recombinant clones were analyzed by nucleotide sequencing. The selected recombinant plasmid (pET28-SP-PmPPAE2 and pET32-WSSV453) was then transformed into E. coli Rosetta (DE3)-pLysS cells (Novagen) for recombinant protein expression and induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside. At 6 h after induction, E. coli cells were harvested by centrifugation at 8,000 rpm for 15 min. The pellets were resuspended in 20 mM Tris-HCl (pH 8.0) and disrupted by an ultrasonic oscillator. The rSP-PmPPAE2 and rWSSV453, as a thioredoxin (trx) fusion protein (rWSSV453-trx), were each purified and refolded as described previously (35). In addition, the WSSV453 PCR product was digested with EcoRI and XhoI and cloned into the EcoRI/XhoI sites of the pET43.1a expression vector (Novagen) containing the NUS fusion protein. The selected recombinant plasmid (pET43.1a-WSSV453) was then transformed into E. coli Rosetta (DE3)-pLysS cells (Novagen), and the expression of rWSSV453, as a NUS fusion protein (rWSSV453-NUS), was induced as described above. The recombinant protein preparations were evaluated for purity by SDS-PAGE analysis and visualized by staining with Coomassie Blue. The concentration of the recombinant proteins was quantified by the Bradford assay.
In addition, the functionally active PmPPAE2 was produced. In this study, the activation site of PmPPAE2 was changed to allow activation by factor Xa. An expression construct of the full-length PmPPAE2 was generated by amplifying the coding sequence with the specific primer pairs PmPPAE2SpeI/ PmPPAE2XbaI (Table 1) from shrimp, P. monodon, cDNA and cloning into the vector pIZT/V5-His (Invitrogen). The resulting construct (pIZT-PmproPPAE2) plasmid, after sequence confirmation, was used as the template to produce mutant plasmid according to PCR base mutagenesis. A mutation was introduced to change the codon at the predicted activation site of PmproPPAE2 from NLNK to IEGR (a cleavage site of bovine factor Xa) using specific primer pairs PmPPEA2-IEGR-F/R (Table 1). This construct was named PmproPPAE2 Xa . After DNA sequence verification, the plasmids were used to transfect into S2 cells using Cellfectin. At 48 h post-transfection, the cell culture medium was collected, and the cells were removed by centrifugation. The supernatant was then concentrated using a 10-kDa cutoff filter (Millipore). Proteins were analyzed by SDS-PAGE and visualized by staining with Coomassie Blue and immunoblotting. Then the proteolytic activation of rPmproPPAE2 Xa by factor Xa was confirmed (data not shown).
Co-immunoprecipitation-The binding of WSSV453 and PmPPAE2 proteins was confirmed using a Pierce co-immunoprecipitation kit according to the manufacturer's instructions. Amino-link plus coupling resin and affinity-purified antibody of rSP-PmPPAE2 protein were incubated in a spin column at room temperature for 3 h and then washed in 1 M NaCl to remove the unbound antibody. The purified rWSSV453-trx protein (10 M) or trx protein (10 M) was incubated with rSP-PmPPAE2 protein (10 M) in spin column containing affinitypurified antibody at 4°C for 16 h. Bound proteins were eluted and analyzed by SDS-PAGE with Coomassie Blue staining and immunoblotting. For immunoblotting, proteins were transferred to a nitrocellulose membrane (Amersham Biosciences Hybond ECL nitrocellulose membrane; GE Healthcare) after electrophoresis. Membranes were blocked by incubation in Tris-buffered solution (137 mM NaCl, 3 mM KCl, 25 mM Tris-HCl, pH 7.6) containing 0.05% (v/v) Tween 20 (TBST) and 1.5% (w/v) skimmed milk powder at room temperature for 2 h, then probed with mouse anti-His tag monoclonal antibody (1:3,000; GE Healthcare) in Tris-buffered solution, and washed twice in TBST. Primary antibodies were detected using alkaline phosphatase-conjugated rabbit antimouse IgG (1:10,000; Sigma) and visualized using incubation in bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as the chromogenic substrate.
Determination of the Proteinase Activity in PmPPAE2silenced and WSSV-infected Shrimp-The proteinase activity of PmPPAE2-silenced and WSSV-infected shrimp was compared so as to investigate the corresponding serine proteinase activity and thus whether WSSV infection suppresses the PO activation via inhibition of PmPPAE2 in the shrimp proPO system. Shrimp were divided into two groups. In the first group, shrimp were divided into three subgroups that were injected with PmPPAE2 dsRNA (experimental group) or with either GFP dsRNA or SSS (control groups). The PmPPAE2 dsRNA was generated in vitro using the T7 RiboMAX TM Express large scale RNA production system (Promega) with the T7PmPPAE2i-F/-R and PmPPAE2i-F/-R (Table 1) gene-specific primer pairs as reported (24). Juvenile shrimp were intramuscularly injected with PmPPAE2 dsRNA (2.5 g/g shrimp, wet body weight) or control GFP dsRNA (same concentration) or SSS. At 24 hpi, shrimp were injected with dsRNA together with 1 g/g shrimp wet body weight of each of LPS and laminarin and then reared for a further 48 h prior to determination of the proteinase activity. The second group of shrimp were divided into two subgroups and injected with either WSSV (experimental subgroup) or PBS (control subgroup) and then reared for a further 48 h prior to assay for proteinase activity. Each group was tested in triplicate using three shrimp per replication. Evaluation of the proteinase activity was performed by incubation of shrimp hemolymph protein (250 g) in 100 l of Tris-HCl (50 mM, pH 8.0) with three different kinds of proteinase substrates that have been reported previously to be suitable for the proteinases involved in the insect proPO system (36) Functional Analysis of WSSV453 by RNAi-In vivo WSSV453 gene silencing, as well as double silencing of WSSV453 and PmPPAE2 genes, during WSSV infection was performed. The WSSV453 dsRNA and PmPPAE2 dsRNA were generated in vitro using the specific T7WSSV453i-F/-R and WSSV453i-F/-R, as well as T7PmPPAE2i-F/-R and PmPPAE2i-F/-R primer pairs (Table 1). Juvenile shrimp were intramuscularly injected with 25 l of SSS containing either (i) WSSV453 dsRNA (5 g/g shrimp, wet body weight) or (ii) WSSV453 and PmPPAE2 dsRNA (5 g each/g shrimp, wet body weight). Three control groups were injected with (iii) PmPPAE2 dsRNA or (iv) GFP dsRNA (same concentration) or (v) with SSS. Shrimp were then injected at 3 hpi with WSSV (LD 50 dose) and reared for a further 48 h before harvesting the hemolymph to determine the efficiency of the WSSV453 dsRNA gene knockdown. The hemocyte total RNA was extracted from the WSSV453 knockdown and WSSV453-PmPPAE2 knockdown and two the control shrimp groups (PmPPAE2 dsRNA and GFP dsRNA-injected) and analyzed by two stage semiquantitative RT-PCR using the WSSV453i-F/-R primers and PPAE2RT-F/R (Table 1) as described above, including the use of the EF1-␣ fragment as an internal control for cDNA template normalization and the VP28 primer to determine the effect of WSSV453 gene silencing on WSSV replication. In addition, the hemolymph of WSSV453 knockdown and WSSV453-PmPPAE2 knockdown shrimp and control shrimp were collected at 48 hpi with WSSV, and their PO activity was determined as described above.
Effect of WSSV453 on Shrimp PO Activity-The PO activity inhibitory activity of WSSV453 via inhibition of PmPPAE2 activity was evaluated. rPmproPPAE2 Xa (2.3 M) were preincubated with rWSSV453-NUS (or rNUS as control) (23 M) in 25 l of 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl 2 (pH 8.0) for 1 h, and each protein was incubated in separate wells of a 96-well plate (costar). Then factor Xa (0.25 g) was added to activate PmPPAE2 activity and incubate for 1 h, and then the total plasma protein of shrimp (0.5 mg of protein) was added into each reaction and incubated for 1 h followed by adding tyrosine (50 l of 1.5 mg/ml in water; Sigma) as substrate for determining PO activity, and 10 mM Tris-HCl (pH 8.0) up to 150 l was added. The PO activity was observed by measuring the absorbance at 470 nm at 6 h using the plate reader (Spec-traMax M5). All experiments were performed in replicate. Statistical analysis was performed by analysis of variance followed by Duncan's test.

Cumulative Mortality of proPOs-silenced Shrimp after WSSV
Infection-To investigate the potential important role of PmproPO1 and PmproPO2 in response to WSSV infection, suppression of both the PmproPO1 and PmproPO2 genes was performed by dsRNA-mediated gene silencing. The hemolymph from three survival shrimp in each group were collected at 2 dpi to determine the efficiency of gene silencing of PmproPO1/2. Both PmproPO transcripts were effectively knocked down to an undetected level by the two-stage RT-PCR (Fig. 1A). Then the cumulative mortality of the PmproPO1/2 co-silenced shrimp after WSSV infection were ascertained and compared with that of the GFP dsRNA-injected and SSS-injected control shrimp. The proPO1/2 co-silenced shrimp exhibited a higher mortality rate than the control groups during the 1-3-dpi period (Fig. 1B), reaching 75 and 95% mortality by 2 and 3 dpi compared with 33% and 63-78% for the control groups at 2 and 3 dpi, respectively. The result indicated that the shrimp proPO system is important in the shrimp defense against WSSV infection. When the proPO system was suppressed, shrimp were more susceptible to WSSV infection, especially at the early phase of WSSV infection. However, the cumulative mortality of the proPOs-silenced shrimp and control groups reached 100% at 4 and 4 -5 dpi, respectively. Antiviral Effect of the Reactive Compounds Generated by the proPO Cascade-The toxicity of the melanization reaction to WSSV and its ability to neutralize WSSV infection was investigated. The WSSV suspension was incubated with the melanization reaction (HLS and dopamine), prior to injection into the shrimp, and then the shrimp survival rate was monitored over 10 dpi, along with that for the control shrimp that were injected with WSSV incubated with either (i) HLS or (ii) dopamine and those injected with (iii) dopamine ϩ HLS or (iv) WSSV only. A much higher shrimp survival rate was found in the group that was injected with the melanization reaction-treated WSSV compared with that in the other control groups (Fig. 2A). Indeed, the survival rate was maintained at over 80% up to the end of the assay (10 dpi), whereas the survival rate of the control shrimp declined to less than 40 and 20% at 4 and 5 dpi and to 0% at 6 -9 dpi. In addition, VP28 transcripts were not detected in shrimp that were injected with the melanization reactiontreated WSSV, whereas they were detected in all the control groups (Fig. 2B). This result demonstrated that the melanization reaction products generated through the proPO system could neutralize the WSSV infections in shrimp.
PO Activity of WSSV-infected Shrimp-Because melanization in P. monodon appears to play an important role in the antiviral response, it was of interest that it has been reported in insects that some viruses are capable of suppressing the host melanization (31). To determine the effect of WSSV infection on the melanization cascade in P. monodon, the PO activity in the hemolymph from WSSV-infected shrimp was ascertained at 1, 2, and 3 dpi. The PO activity of WSSV-infected shrimp was dramatically (ϳ6-fold) and significantly decreased at 2 and 3 dpi compared with that of the uninfected shrimp (Fig. 3A). Furthermore, the addition of trypsin (exogenous proteinase) into the PO reaction of the WSSVinfected and PBS-injected shrimp hemolymph restored the PO activity of the WSSV-infected shrimp, and no significant difference in the PO activity was then observed between the infected and uninfected shrimp (Fig. 3B). Thus, the shrimp PO activity might be suppressed by WSSV via the inhibition  of the serine proteinase activity (trypsin-like serine proteinase) of the proPO cascade.
Identification of Potential WSSV Proteins That Regulate the proPO Cascade during WSSV Infection Using the Yeast Twohybrid Assay-To identify WSSV proteins that might inhibit the proteinase activity in the shrimp proPO cascade, the yeast two-hybrid screening of a WSSV library for viral proteins that could interact with PmPPAE2 was performed. Only one positive clone was identified among the ϳ2 ϫ 10 5 clones that were screened. The prey plasmid from the positive colony was then extracted. The interaction between PmPPAE2 and the isolated prey plasmid was reconfirmed by co-transformation into yeast and analysis in the two-hybrid system. Co-transformation of PmPPAE2/pGBKT7 and the empty pGADT7 plasmid was included as a negative control. Interaction indicated by the blue colony growth on SD/ϪL/ϪW/ϪA/ϪH/X-␣-gal-selective medium was observed from yeast containing PmPPAE2 and the isolated prey plasmid but not from the control yeast colonies (Fig. 4A). Sequencing and comparison of the deduced amino acid sequences of this clone with the GenBank TM database using the BLAST program revealed that it was WSSV453 (100% identity, GenBank TM accession number AAL89321.1). This 306-bp ORF of WSSV453 has been reported in the nucleotide sequence of the Thai WSSV strain and encodings for a predicted 101-amino acid protein with a calculated molecular mass of 11.92 kDa and an estimated pI of 9.95. WSSV453 is an uncharacterized protein with no putative domains being identified by the SMART program and database searching. WSSV453 is identical (100% amino acid sequence identity) to the gene of Taiwan WSSV strain WSV394 (GenBank TM accession number NP477916).
Interaction of rWSSV453 and rPmPPAE2 Protein by Coimmunoprecipitation-To confirm the interaction of WSSV453 and PmPPAE2 proteins, the rWSSV453 and the SP domain of PmPPAE2 (rSP-PmPPAE2) were separately expressed in E. coli. However, the production of a stable rWSSV453 could not be obtained because the recombinant protein degraded rapidly (data not shown). Therefore, the rWSSV453-thioredoxin fusion protein (rWSSV453-trx) was produced and used in the experiments. Co-immunoprecipitation, performed using an anti-His antibody according to the Western blotting analysis technique, revealed a positive binding between rWSSV453-trx and rSP-PmPPAE2 (Fig. 4B), as shown by the presence of both of these protein bands in the elution step compared with only the rSP-PmPPAE2 band in the control (trx) elution (Fig. 4C). These results supported that WSSV453 binds to PmPPAE2.
Serine Proteinase Activity in WSSV-infected Shrimp and in PmPPAE2 Knockdown Shrimp-The proteinase activity of WSSV-infected shrimp was further examined in the hemolymph and compared with the control shrimp injected with PBS using different proteinase substrates (Fig. 5, A-C). The highest reduction in the proteinase activity in WSSV-infected shrimp compared with the control PBS-injected shrimp (68.6%) was found with the B-6388 proteinase substrate. This reduction corresponded to the decreased proteinase activity found in the PmPPAE2 knockdown shrimp (Fig. 5D). The results suggested that WSSV suppresses the proPO system via inhibition of the proteinase cascade and that PmPPAE2 is likely to be a target enzyme that is suppressed by the virus.
Functional Analysis of WSSV453 by RNAi-To investigate the function of WSSV453 in the suppression of the shrimp PO activity, WSSV453 or both WSSV453 and PmPPAE2 transcription were silenced by RNAi, and the PO activity of those groups after WSSV infection was determined. The efficiency of the gene knockdown was determined by semiquantitative RT-PCR analysis, which showed that the WSSV453 and PmPPAE2 transcript levels were significantly decreased in the WSSV453-silenced shrimp and PmPPAE2-silenced shrimp, respectively, whereas injection of the GFP dsRNA (control) had no significant effect on PmPPAE2 and WSSV453 transcription level (Fig.  6A). Moreover, the gene silencing of WSSV453, as well as PmPPAE2, had no effect on the replication of WSSV, as shown by the similar VP28 transcription levels in control shrimp (Fig.  6A). A significant increase in the total hemolymph PO activity was observed in the WSSV453 knockdown shrimp compared with the control group, but the double silencing of . Interaction of PmPPAE2 and WSSV453 proteins. A, yeast two-hybrid assay, the PmPPAE2 construct was synthesized in-frame with the BD domain of the pGBKT7 plasmid, whereas WSSV453 was in-frame with the AD domain of pGADT7. Results for three independent transformants from SD/ϪL/ϪW plates when grown on SD/ϪL/ϪW/ϪA/ϪH/X-␣-gal plates, where interaction (ϩ) is indicated by blue yeast colonies and no interaction (Ϫ) by no growth. Yeast containing PmPPAE2/pGBKT7 and empty pGADT7 plasmid served as the negative control. B and C, Western blot analysis of the co-immunoprecipitation of WSSV453 (as rWSSV453-trx) with PmPPAE2 (as rSP-PmPPAE2), the purified rSP-PmPPAE2 (10 M) was incubated with rWSSV453-trx protein (B, 10 M) or trx protein (C, 10 M) (control protein) and then subjected to co-immunoprecipitation (CO-IP) with anti-SP-PmPPAE2 agarose beads. The proteins were separated by SDS-PAGE and analyzed by Western blotting with anti-His antibody.
PmPPAE2 and WSSV453 did not increase the PO activity during WSSV infection. Thus, gene knockdown of WSSV453 could restore the PO activity dependent on PmPPAE2 in WSSV-infected shrimp, suggesting that WSSV453 might function in suppression of PO activity during WSSV infection via interaction with PmPPAE2. However, the increased PO activity in the WSSV453 knockdown shrimp was still below that in the control SSS-injected shrimp (Fig. 6B), suggesting that other unknown mechanism(s) or other WSSV protein(s) might also be involved in the suppression of the cascade during WSSV infection.
Effect of WSSV453 on Shrimp PO Activity-The recombinant protein of active PmproPPAE2 Xa was produced with an estimated molecular mass of 43 kDa. The proteolytic activation of rPmproPPAE2 Xa by factor Xa was confirmed (data not shown). In addition, rWSSV453 was expressed in E. coli as a fusion protein with a NUS tag to increase the protein solubility. The rWSSV453-NUS and rNUS (control) proteins have estimated molecular masses of 72 and 64 kDa, respectively. The rWSSV453-NUS was preincubated with PmproPPAE2 Xa protein and followed by adding factor Xa and shrimp plasma, and then PO activity was measured by adding tyrosine as substrate. The results showed a significant increase of PO activity in shrimp plasma (ϳ65%) by adding activated PmproPPAE2 Xa protein (Fig.  7) compared with the control group (nonactivated plasma by PmPPAE2 Xa ), but the preincubation of PmproPPAE2 Xa with rWSSV453-NUS protein showed the significant decrease (ϳ40%) of PO activity when compared with preincubation with NUS as control protein (Fig. 7). This indicated that rWSSV453-NUS has the ability to inhibit PO activation probably via inhibition of PmP-PAE2 activity in shrimp plasma.

DISCUSSION
Melanization, activated through the proPO-activating system, participates in invertebrate innate immune responses and appears to play a key role in the non-self-recognition system being responsible for parasite entrapment and microbe killing, as well as wound healing (8,17,(37)(38)(39)(40). The reactive intermediates produced during melanin synthesis, such as reactive oxygen and nitrogen intermediates and quinone-like substances, are toxic to some microorganisms and multicellular parasites and so restrain the invasion of these pathogens into the host body cavity (8,(17)(18)(19)(20)(21)40). The complex melanization cascade involves several proPO-associated proteins that are fairly well characterized in many invertebrates (8,38,39). In P. monodon, two proPO isoforms (PmproPO1 and PmproPO2) and two proPO-activating enzymes (PmPPAE1 and PmPPAE2) have been identified and demonstrated to function in the shrimp proPO system and to play a crucial role in the defense against Vibrio harveyi and Fusarium solani infection (22)(23)(24)(25). The melanization reaction products of shrimp also exhibited antimicrobial effects toward shrimp major bacterial and fungal pathogens (25). Here, we show the importance of the P. monodon melanization reaction in the defense against WSSV. In vivo gene co-silencing of PmproPO1 and PmproPO2 followed by WSSV infection resulted in a higher mortality rate of shrimp at 1-3 dpi compared with the control shrimp (Fig. 1), implicating the role of proPO system in defense against virus infection. Additionally, P. monodon melanization reaction products exhibited an in vitro neutralization effect on WSSV infectivity, as shown by the reduced shrimp mortality and VP28 transcript levels when infected with WSSV preincubated with a melanization reaction prior to injection into the shrimp ( Fig.  2A). This could reflect that the shrimp melanization cascade generates several highly reactive and toxic compounds (18 -21) that are toxic to WSSV, because no viral VP28 transcript expression was observed in these shrimp (Fig. 2B). Likewise in Manduca sexta, the proPO activation and PO-generated reactive compounds, such as 5,6-dihydroxyindole, was reported to exhibit broad spectrum antibacterial, antifungal and antiviral activities (18,19). Taken together, these results suggest that P. monodon melanization is an important immune response to virus infection.
In addition, WSSV infection affected the shrimp proPO system by inhibiting the PO activation. The PO activity of WSSVinfected shrimp had a reduced PO activity at 2 and 3 dpi compared with the uninfected shrimp (Fig. 3A). Interestingly, the exogenous addition of trypsin into the PO reaction of the hemolymph from WSSV-infected shrimp resulted in the full restoration of PO activity (Fig. 3B), suggesting that WSSV might block the PO activation via inhibition of a serine proteinase (trypsin-like serine proteinase) in the shrimp proPO cascade.
Similarly, in Pacifastacus leniusculus, it was found that WSSV inhibits the proPO system upstream of PO because no melanization was found in the granular cells of WSSV-infected crayfish, but the HLS PO activities of both sham-and WSSVinjected crayfish were the same, as were the proPO transcript levels detected by RT-PCR (41). In line with these results, it was previously reported that various kinds of pathogens suppress the host proPO system to obtain a successful infection. For example, the virulent bacterium Photorhabdus produces a small molecule antibiotic, (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene, to inhibit PO in the insect host M. sexta. Mutation of this gene to give a non-PO inhibitor isoform also resulted in the bacteria being nonvirulent, suggesting that PO activity is required for the elimination of this bacterium (42). In addition, the parasitic wasp M. demolitor that oviposits FIGURE 6. In vivo gene silencing of WSSV453 and co-silencing of WSSV453 and PmPPAE2 by RNAi. Shrimp were injected with WSSV453 dsRNA (5 g/g shrimp) or with WSSV453 and PmPPAE2 dsRNA (5 g each/g shrimp). Three control groups were injected with PmPPAE2 dsRNA (5 g/g shrimp) or GFP dsRNA (5 g/g shrimp) or SSS (controls). At 3 hpi, the shrimp were injected with WSSV and then reared for a further 48 h before collecting the hemolymph to ascertain the hemocyte WSSV453 or PmPPAE2 or VP28 transcript level and hemolymph PO activity. A, the efficiency of dsRNA-mediated gene silencing of WSSV453 or PmPPAE2 transcripts at 2 dpi was examined by RT-PCR. EF1-␣ was used as the internal reference gene for RT-PCR. In the shown gel, the lane for each condition represents the results from individual shrimp. B, the effect of WSSV453 gene knockdown on the PO activity of WSSV-infected shrimp. The total hemolymph PO activity was defined as ⌬A 490 /mg of total protein per min. The data are shown as the means Ϯ S.D. (error bars) and are derived from three independently replicated experiments. Different letters (above each bar) indicate a significant difference (p Ͻ 0.05) in the mean PO activity among the six samples.
in M. sexta larvae also injects M. demolitor bracovirus, MdBV (31). This virus produces serine proteinase inhibitors (Egf1.0 and Egf1.5) that block the processing and the amidolytic activity of the host proPO activating proteinases (pro-PAP1 and 3), whereas Egf1.0 also binds to proPO and the serine proteinase homologs 1 and 2 (SPH1 and 2). Thus, Egf is important to prevent the host proPO activation and so the PO activity. If the Egf1.0 or Egf1.5 gene is inactive, the wasp eggs are melanized and fail to survive in the host (32,33).
Interestingly, using the yeast two-hybrid screening, we found that the WSSV protein WSSV453 (AAL89321.1) interacts with PmPPAE2 (Fig. 4A), one of the serine proteinase enzymes of the P. monodon proPO system. Co-immunoprecipitation confirmed that PmPPAE2 directly interacts with WSSV453 (Fig.  4B). WSSV453 is a small viral protein (estimated molecular mass of 11.92 kDa) but is of unknown function with no functional domains being predicted by SMART analysis. PmPPAE2 is a terminal clip-serine proteinase (clip-SP) that is implicated in the proPO activation cascade of shrimp converting the proPO to active PO. PmPPAE2 contains an N-terminal clip domain and a C-terminal trypsin-like SP domain that has a catalytic triad comprised of three catalytic residues (His, Asp, and Ser) in the SP domain (24). Because PmPPAE2 plays a very important role in proPO activation and WSSV453 shows a binding activity to PmPPAE2, we speculated that binding of WSSV453 with PmPPAE2 might somehow interfere with proPO activation in P. monodon. In the same way, the reduced proteinase activity in WSSV-infected shrimp at 48 hpi supports that the WSSV-inhibited PO activity was mediated via inhibition of proteinase activity (Fig. 5, A-C). Moreover, using different proteinase-specific substrates the same pattern of reduced proteinase activity was found in both WSSV-infected and PmP-PAE2-silenced shrimp, especially when using B-6388 as substrate (Fig. 5D). This implies that PmPPAE2 may be one of the inhibited proteinase enzyme targets during WSSV infection.
The increased PO activity in WSSV453-silenced shrimp after WSSV infection at 48 hpi but not in double silencing of WSSV453 and PmPPAE2 gene (Fig. 6) suggested that WSSV453 might be involved in the inhibition of proPO activation in the P. monodon hemolymph via PmPPAE2. Moreover, we found that PO activity of shrimp plasma was significantly activated after adding active PmproPPAE2 Xa (activated by factor Xa), but the PO activity was significantly decreased when preincubating PmproPPAE2 Xa with rWSSV453-NUS protein (Fig. 7). This again supports the ability of WSSV453 in interfering PO activation in shrimp plasma via binding to PmPPAE2 protein. However, these results do not fully reveal how WSSV453 impaired proPO activation via PmPPAE2.
Generally, the proPO activating enzyme(s) in proPO systems in many species are regulated by serine proteinase inhibitors (serpins) (43,44). The nucleotide sequence of WSSV453 shows no homology to other known serpins, suggesting that this viral protein may have a different mechanism for disabling the host melanization. Dengue virus, which is transmitted by the mosquitoes Aedes aegypti and Aedes albopictus, encodes for the NS1 protein that can interfere with the host coagulation and contributes to the hemorrhage in Dengue hemorrhagic fever. The secreted Dengue virus NS1 binds to thrombin and prothrombin, which play a very important role in the activation of coagulation, but NS1 did not decrease thrombin activity (45). Thus, rNS1 could inhibit prothrombin activation and prolong the activated partial thromboplastin time in Dengue hemorrhagic fever patients (45). By analogy, one possibility is that the binding of WSSV453 to PmPPAE2 might interfere with the PmproPPAE2 activation and so prevent PmPPAE2 from activating proPO to PO.
In summary, the importance of the proPO system of P. monodon in the defense against WSSV infection was established, as was the fact that WSSV could suppress the host proPO system. In addition, the possible interaction of PmPPAE2 of the proPO system with the WSSV453 protein might result in the suppression of the host PO activity. These findings provide novel insights into the molecular events of virus-host interactions directed by melanization in shrimp and reveal a novel regulatory function of WSSV453. Nevertheless, the biological function of WSSV453 in the shrimp proPO system remains to be fully elucidated.