WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity.

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus).

Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could be useful in terms of protective antigens for vaccine development. This study aims to evaluate the potential use of Tdr protein as a novel subunit vaccine against A. veronii infection in Nile tilapia. The Tdr gene from A. veronii was cloned into the pET28b expression vector, and the recombinant protein was subsequently produced in Escherichia coli strain BL21 (DE3). Tdr was expressed as an insoluble protein and purified by affinity chromatography. Antigenicity test indicated that this protein was recognized by serum from A. veronii infected fish. When Nile tilapia were immunized with the Tdr protein, specific antibody levels increased significantly (p-value <0.05) at 7 days post-immunization (dpi), and peaked at 21 dpi compared to antibody levels at 0 dpi. Furthermore, bacterial agglutination activity was observed in the fish serum immunized with the Tdr protein, indicating that specific antibodies in the serum can detect Tdr on the bacterial cell surface. These results suggest that Tdr protein has potential as a vaccine candidate. However, challenging tests with A.veronii in Nile tilapia needs to be investigated to thoroughly evaluate its protective efficacy for future applications.

Synthetic peptides derived from predicted B cell epitopes of nervous necrosis virus (NNV) show antigenicity and elicit immunogenic responses in Asian seabass (Lates calcarifer).

Nervous necrosis virus (NNV) has spread throughout the world, affecting more than 120 freshwater and marine fish species. While vaccination effectively prevents disease outbreaks, the difficulty of producing sufficient viruses using cell lines continues to be a significant disadvantage for producing inactivated vaccines. This study, therefore, explored the application of synthetic peptides as potential vaccine candidates for the prevention of NNV in Asian seabass (Lates calcarifer). Using the epitope prediction tool and molecular docking, three predicted immunogenic B cell epitopes (30-32 aa) derived from NNV coat protein were selected and synthesised, corresponding to amino acid positions 5 to 34 (P1), 133 to 162 (P2) and 181 to 212 (P3). All the predicted peptides interact with Asian sea bass's MHC class II by docking. The antigenicity of these peptides was determined through ELISA and all peptides were able to react with NNV-specific antibodies. Subsequently, the immunogenicity of these synthetic peptides was investigated by immunisation of Asian seabass with individual peptides (30 µg/fish) and a peptide cocktail (P1+P2+P3, 10 µg each/fish) by intraperitoneal injection, followed by a booster dose at day 28 post-primary immunisation. There was a subset of immunised fish that were able to induce upregulation of immune genes (IL-1ß, TNFα, MHCI, MHCII ß, CD4, CD8, and IgM-like) in the head kidney and spleen post immunization. Importantly, antibodies derived from fish immunised with synthetic peptides reacted with whole NNV virions, and sera from P1 group could neutralise NNV in an in vitro assay. Taken together, these findings indicate that synthetic linear peptides based on predicted B cell epitopes exhibited both antigenic and immunogenic properties, suggesting that they could be potential vaccine candidates for the prevention of NNV in fish.

Immunological response of 35 and 42 days old Asian seabass (Lates calcarifer, Bloch 1790) fry following immersion immunization with Streptococcus iniae heat-killed vaccine.

Early disease prevention by vaccination requires understanding when fry fish develop specific immunity to a given pathogen. In this research, we explored the immune responses of Asian seabass (Lates calcarifer) at the stages of 35- and 42- days post-hatching (dph) to an immersive heat-killed Streptococcus iniae (Si) vaccine to determine whether fish can produce specific antibodies against the pathogen. The vaccinated fish of each stage (V35 and V42) were immersed with the Si vaccine at 107 CFU/ml for 3 h, whereas the control groups (C35 and C42) were immersed with tryptic soy broth (TSB) in the same manner. Specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) before and post-immunization (i.e., 0, 7, and 14 days post-immunization, dpi). Expression of innate (TNFα and IL-1ß) and adaptive (MHCI, MHCII, CD4, CD8, IgM-like, IgT-like, and IgD-like) immune-related genes were evaluated at the same time points with the addition of 1 dpi. The results showed that a subset of immunized fish from both V35 and V42 fry could elicit specific antibodies (IgM) against Si at 14 dpi. All tested innate and adaptive immune genes upregulated at 7 dpi among fish in V35 group. Interestingly, 42 dph fish appeared to respond to the Si vaccine faster than that of 35 dph, as a significant increase in transcripts was observed in CD4, IL-1ß, IgM-like, and IgD-like at 1 dpi; and specific antibody titers of some fish, although not all, were higher than a threshold (p = 0.05) since 7 dpi. In conclusion, this study reveals that 35-42 dph Asian seabass fry can elicit specific immunity to Si immersion vaccine, suggesting that early vaccination of 35 dph fry Asian seabass is feasible.

Refolded recombinant major capsid protein (MCP) from Infectious Spleen and Kidney Necrosis Virus (ISKNV) effectively stimulates serum specific antibody and immune related genes response in Nile tilapia (Oreochromis niloticus).

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1ß and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.

Tilapia lake virus (TiLV): Genomic epidemiology and its early origin.

Tilapia lake virus (TiLV) is an emerging virus that is rapidly spreading across the world. Over the past 6 years (2014-2020), TiLV outbreaks had been reported in at least 16 countries, spanning three continents, including Asia, Africa, and America. Despite its enormous economic impact, its origin, evolution and epidemiology are still largely poorly characterized. Here, we report eight TiLV whole-genome sequences from Thailand sampled between 2014 and 2019. Together with publicly available sequences from various regions of the world, we estimated the origin of TiLV to be between 2003 and 2009, 5-10 years before the first report of the virus in Israel in 2014. Our analyses consistently showed that TiLV started to spread in 2000s, and reached its peak in 2014-2016, matching well with the timing of its first report. From 2016 onwards, the global TiLV population declined steadily. This could be a result of herd immunity building up in the fish population, and/or a reflection of a better awareness of the virus coupled with a better and more cautious protocol of Tilapia importation. Despite the fact that we included all publicly available sequences, our analyses revealed long unsampled histories of TiLVs in many countries, especially towards its basal diversification. This result highlights the lack and the need for systematic surveillance of TiLV in fish.

In Vitro Antimicrobial Activity of Piper retrofractum Fruit Extracts against Microbial Pathogens Causing Infections in Human and Animals.

Long pepper (Piper retrofractum Vahl) is a Thai medicinal herb which has been used as one of the common ingredients in variety of Thai foods. Here, we investigated antimicrobial activities of crude bioactive metabolites extracted from fruits of P. retrofractum against 10 pathogenic organisms (bacteria and yeast) causing opportunistic infections in human or animals including Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC2921, Escherichia coli ATCC25922, Klebsiella pneumonia TISTR1843, Pseudomonas aeruginosa ATCC741, Salmonella typhi (clinical isolate), Vibrio parahaemolyticus (XN98 and 5HP), and Candida albicans ATCC90020. The results of disk diffusion test showed that the extract from methanol solvent exhibited greater antibacterial activity than other solvents with inhibition zones ranging from 0.5 to 8.0 mm, respectively. Subsequently, minimal inhibition concentration (MIC) determined by the colorimetric assay confirmed that methanol extracts showed consistent results with disk diffusion method. In summary, in vitro assays suggest that methanol is the best solvent for extraction of bioactive metabolites from P. retrofractum fruits. This crude extract can inhibit the majority of human and animal pathogens. This opens up a potential use of pepper fruits in prevention of food-contaminating microorganisms.

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade.

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

The interaction between PmHtrA2 and PmIAP and its effect on the activity of Pm caspase.

Apoptosis is an essential mechanism in multicellular organisms which results in the induction of cell death. Important apoptotic proteins, including high temperature requirement A2 (PmHtrA2; also known as serine protease), inhibitor of apoptosis protein (PmIAP) and Pm caspase, have been previously identified in black tiger shrimp, Penaeus monodon. However, the relevance among these proteins in apoptosis regulation has not been established yet in shrimp. Here, we showed that PmHtrA2 was able to interact with PmIAP and the binding of the two proteins was mediated by the BIR2 domain of PmIAP. In addition, the BIR2 of PmIAP was shown to be able to inhibit Pm caspase activity. The inhibitory effect of the BIR2 domain on Pm caspase was impaired under the presence of the IBM peptide of PmHtrA2, implying a role for PmHtrA2 in apoptosis activation. Our combined results suggested that P. monodon possesses a conserved mechanism by which the caspase-3 activity is modulated by HtrA2 and IAP, as previously seen in insects and mammals.

A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp.

BACKGROUND: RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called "multi-WSSV dsRNA." A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners. RESULTS: For a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (µg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge. CONCLUSION: The present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.

Laminin receptor protein is implicated in hemocyte homeostasis for the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp Penaeus (Litopenaeus) vannamei (Pv) caused a dramatic reduction specifically in hyaline hemocytes prior to death. Since apoptosis was not detected in hemocytes or hematopoietic cells, other possible causes of hemocyte loss were investigated. Reports that suppression of crustacean hematopoietic factor (CHF)-like protein or hemocyte homeostasis-associated protein (HHAP) also reduced shrimp hemocyte counts led us to carry out yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays to test for interactions between Lamr and Pv homologues to these proteins (PvCHF-like and PvHHAP). The assays revealed that Lamr bound to both these homologues, but that the homologues did not bind to each other. Subsequent RT-PCR assays confirmed that PvLamr dsRNA injection significantly reduced expression levels for both PvCHF-like and PvHHAP genes. Further work is needed to determine how interaction among these three proteins can regulate shrimp hemocyte homeostasis.

Two new anti-apoptotic proteins of white spot syndrome virus that bind to an effector caspase (PmCasp) of the giant tiger shrimp Penaeus (Penaeus) monodon.

White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55-214) of Penaeus monodon caspase (PmCasp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length PmCasp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with PmCasp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon (Pm caspase) did not block apoptosis induced by PmCasp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon.

Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Construction and application of a protein interaction map for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

Co-interactive DNA-binding between a novel, immunophilin-like shrimp protein and VP15 nucleocapsid protein of white spot syndrome virus.

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly.