p53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

Co-Infection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in the Wild Crustaceans of Andaman and Nicobar Archipelago, India.

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.

The role of Astakine in Scylla paramamosain against Vibrio alginolyticus and white spot syndrome virus infection.

Astakine is a crucial factor in the proliferation and differentiation of hematopoietic stem cells and is directly involved in hematopoiesis in crustaceans. To assess the role of Astakine in the innate immune system of Scylla paramamosain, the immune responses in healthy and Astakine-inhibited S. paramamosain were investigated in the present study. The RNA transcripts of Astakine were widely distributed in all examined tissues, with significantly higher levels of expression in hemocytes of both healthy and challenged S. paramamosain with Vibrio alginolyticus and WSSV. When Astakine was knocked down by RNA interference technology, immune-related genes, including Janus kinase, prophenoloxidase, hemocyanin, ß-actin, myosin II essential light chain-like protein, signal transducer and activator of transcription, Relish, and C-type-lectin, were significantly down-regulated in hemocytes. The levels of phenoloxidaseactivity (PO), total hemocyte counts (THC) and hemocyte proliferation decreased significantly in hemocytes of Astakine-dsRNA treated S. paramamosain. After being challenged with V. alginolyticus and WSSV, the THC decreased significantly and the levels of hemocyte apoptosis increased significantly in Astakine-dsRNA treated S. paramamosain in comparison with those in infected groups without Astakine-dsRNA treatment. After being challenged with WSSV, the WSSV copies were significantly lower in Astakine-dsRNA treated groups than those in the WSSV infection group, which suggested that knockdown of Astakine was not conductive to WSSV replication and this might be associated with the decreasing THC. The results of survival analysis showed that the survival rate of V. alginolyticus or WSSV infected S. paramamosain decreased significantly following Astakine knockdown. These results suggested that RNA interference of Astakine might weaken the resistance of S. paramamosain to V. alginolyticus or WSSV infection. The weaken resistivity after knockdown Astakine might be related to the changes of important immune-related gene expression, THC, PO activity, proliferation and apoptosis of hemocytes.

SpBcl2 promotes WSSV infection by suppressing apoptotic activity of hemocytes in mud crab, Scylla paramamosain.

White spot syndrome virus (WSSV) is one of the most virulent and widespread pathogens that infect almost all marine crustaceans and therefore cause huge economic losses in aquaculture. The Bcl2 protein plays a key role in the mitochondrial apoptosis pathway, which is a crucial immune response in invertebrates. However, the role of Bcl2 in apoptosis and immunoregulation in mud crab, Scylla paramamosain, is poorly understood. Here, the Bcl2 homolog (SpBcl2) in S. paramamosain was cloned and its role in WSSV infection explored. The expression of SpBcl2 increased at both the transcriptional level and post-transcriptional level after WSSV infection, while the hemocytes apoptosis decreased significantly. Furthermore, there was increase in the level of cytochrome c coupled with an upregulation in the expression of SpBcl2. These results indicated that SpBcl2 suppressed apoptosis by preventing the release of cytochrome c from mitochondria, thereby promoting WSSV replication in mud crab. The findings here therefore provide novel insight into the immune response of mud crabs to WSSV infection.

Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections.

Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

Epigallocatechin-3-gallate inhibit replication of white spot syndrome virus in Scylla paramamosain.

Epigallocatechin-3-gallate (EGCG) has exhibited potential antibacterial and anticancer activities. In this study, we tested whether EGCG can protect the mud crab Scylla paramamosain against white spot syndrome virus (WSSV). Treatment with 1.5 mg/kg EGCG killed crab hemocytes but treatment with 1 mg/kg EGCG did not. Results of the virus challenge experiment confirmed that EGCG could enhance the survival rate of WSSV-challenged crabs, and treatment with 1 mg/kg EGCG significantly increased their resistance to WSSV compared with the control. Thus, 1 mg/kg EGCG was used as the experimental dose in subsequent analyses. EGCG treatment may induce certain immune pathways, such as the phenoloxidase and JAK-STAT pathways, and it also can enhance the activity of prophenoloxidase. EGCG + WSSV treatment significantly reduced the number of WSSV copies at 24, 48, and 72 h post-challenge compared with the control (WSSV challenge without EGCG treatment). These results demonstrate that EGCG may effectively improve innate immunity and survival of WSSV-challenged S. paramamosain and inhibit WSSV replication by blocking the expression of late stage WSSV genes. Therefore, our study presented that EGCG might represent a new potential therapeutic or preventive approach to control white spot syndrome.

Identification and RNA segment assignment of six structural proteins of Scylla serrata reovirus.

Scylla serrata reovirus (SsRV) is one of the most prevalent viral pathogens of the mud crab (S. serrata). The virus represents an unassigned novel genus in the Reoviridae family, and contains 12 double-stranded RNA genomic segments. Previous analysis of virion proteins concluded that SsRV contains at least eight structural proteins, ranging from 25 to 160 kDa. Here, tandem time-of-flight mass spectrometry and Western blotting were used to re-identify the structural proteins. The results indicate that proteins encoded by SsRV segments S1, S3, S6, S9, S11, and S12 are structural proteins.

Identification of mud crab reovirus VP12 and its interaction with the voltage-dependent anion-selective channel protein of mud crab Scylla paramamosain.

Mud crab reovirus (MCRV) is the causative agent of a severe disease in cultured mud crab (Scylla paramamosain), which has caused huge economic losses in China. MCRV is a double-stranded RNA virus with 12 genomic segments. In this paper, SDS-PAGE, mass spectrometry and Western blot analyses revealed that the VP12 protein encoded by S12 gene is a structural protein of MCRV. Immune electron microscopy assay indicated that MCRV VP12 is a component of MCRV outer shell capsid. Yeast two hybrid cDNA library of mud crab was constructed and mud crab voltage-dependent anion-selective channel (mcVDAC) was obtained by MCRV VP12 screening. The full length of mcVDAC was 1180 bp with an open reading frame (ORF) of 849 bp encoding a 282 amino acid protein. The mcVDAC had a constitutive expression pattern in different tissues of mud crab. The interaction between MCRV VP12 and mcVDAC was determined by co-immunoprecipitation assay. The results of this study have provided an insight on the mechanisms of MCRV infection and the interactions between the virus and mud crab.

Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification.

Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 µmol/L F3/B3, 1.6 µmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR.

Tissue distribution of hepatopancreatic parvo-like virus of shrimp in freshwater rice-field crab, Paratelphusa hydrodomous (Herbst).

An attempt was made to determine the replication efficiency of hepatopancreatic parvo-like virus (HPV) of shrimp in different organs of freshwater rice-field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT-PCR, ELISA, Western blot and q-PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large-scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT-PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q-PCR. The results revealed a steady decrease in CT values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post-larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice-field crab could be used as an alternative host for HPV replication and also for large-scale production of HPV.

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab.

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.

High efficacy of white spot syndrome virus replication in tissues of freshwater rice-field crab, Paratelphusa hydrodomous (Herbst).

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice-field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT-PCR, ELISA, Western blot and real-time PCR analyses, and also to use this crab instead of penaeid shrimp for the large-scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT-PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real-time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.

Molecular characterization of eight segments of Scylla serrata reovirus (SsRV) provides the complete genome sequence.

Scylla serrata reovirus (SsRV) is one of the most prevalent viral pathogens of mud crabs (S. serrata). Of the 12 double-stranded RNA (dsRNA) genomic segments (S1-S12), the three largest (S1-S3) and S7 were sequenced previously and were shown to have no or only low sequence homology to known members within the family Reoviridae. The sequences of the remaining segments, S4-S6 and S8-S12, are reported here. With the exception of S4, all have single open reading frames (ORFs) on their positive strands, and the terminal sequences 5'-AUAAA(U)/(C) (A)/(U)…G(A)/(G) (A)/(U) (A)/(C)AAC(G)/(U)AU-3' are conserved among currently and previously sequenced segments. S4 contains two out-of-phase ORFs on the positive strand, suggesting that this segment is bicistronic. The ORFs of segments S4-S6 and S8-S12 have low or no homology to other reovirus genes, with the exception that all of the SsRV segments have high sequence similarity to those of mud crab reovirus (MCRV) and share the same 5'- and 3'-terminal nucleotide sequences, suggesting that the two viruses belong to the same species in the family Reoviridae. Analysis of virion proteins revealed that SsRV contains at least eight structural proteins, with sizes ranging from 25 to 160 kDa.

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions.

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.

Parasites and pathogens of the endosymbiotic pea crab (Pinnotheres pisum) from blue mussels (Mytilus edulis) in England.

A histopathological survey of the commensal pea crab (Pinnotheres pisum) from the mantle cavities of blue mussels (Mytilus edulis) has been conducted. A total of 266 pea crabs from eight sites around the English coastline were examined. Of these, 82 were negative for any visible infections by histology. The remaining pea crabs were infected with an intranuclear bacilliform virus designated as P. pisum bacilliform virus (PpBV) in the hepatopancreatic epithelial cells, peritrichous ciliates on the gills, an intracytoplasmic microsporidian infection of the hepatopancreatocytes, a myophilic microsporidian infection, the gregarine Cephaloidophora fossor in the hepatopancreas, the entoniscid isopod Pinnotherion vermiforme, a low level nematode infection and an acanthocephalan cystacanth. Host reactions to infections were generally subdued. Results are discussed in relation to the endocommensal habitat of the pea crabs.

Monoclonal antibodies produced against VP3 of a novel mud crab dicistrovirus.

Mud crab dicistrovirus (MCDV) is a recently identified single-stranded positive RNA virus, which causes serious economic loss. The three structural proteins of MCDV were separated by SDS-PAGE. In this study, a recombinant MCDV-VP3 was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, three mouse hybridomas (1G6H9, 1E7B8, 4B6E10) producing MAbs against MCDV-VP3 were established. The MAbs obtained were fully characterized using indirect ELISA and Western blot analysis. The ELISA results showed that the titers of MAbs were in the range of 1:3.200×10(3) in culture fluids and 1:2.048×10(5) in ascitic fluids. Using Western blot analyses, we observed the specific characteristic band that defines VP3. It demonstrated that all the MAbs were directed against MCDV-VP3. The results of immunogold transmission electron microscopy (IEM) suggested that MCDV-VP3 is the capsid protein of MCDV. The preparation of MAbs specific to the structure protein of MCDV would be useful for studying the function of the structure proteins and the mechanism of infection and pathogenesis of MCDV.

Cancer pagurus bacilliform virus (CpBV) infecting juvenile European edible crabs C. pagurus from UK waters.

This study provides the first description of an intranuclear bacilliform virus infecting the hepatopancreatic epithelial cells of juvenile European edible crab Cancer pagurus from the English Channel, UK. This is the first field report of a virus infecting a member of the Cancer genus and follows a report of a different virus infection detected in C. pagurus held under experimental conditions. We have named the virus Cancer pagurus bacilliform virus (CpBV). The morphology and size of the nucleocapsid and the complete virion align the virus most closely with intranuclear bacilliform viruses reported from other decapod species. The virus was only observed in juvenile C. pagurus with carapace widths ranging between 20 and 70 mm. Viral infection was not observed in crabs above the minimum landing size of 140 mm carapace width. Due to the importance of C. pagurus as a fisheries target, the increasing relative reliance on crustacean fisheries in Europe and the relative dearth of information on diseases of this species, it is pertinent to consider the effects that infections such as CpBV may have on mortality of juvenile crabs and on the sustainability of the fishery as a whole.

Construction of a gene probe for detection of P virus (Reoviridae) in a marine decapod.

The construction is described of a molecular probe to P virus, a double stranded RNA virus belonging to the Reoviridiae, which is an endemic pathogen of swimming crabs in British coastal waters and the Mediterranean. The probe hybridises to the P virus genome and can be easily produced in large quantities by PCR. It may be used by dot blotting or in situ hybridisation to specifically detect P virus in tissues and cells of natural or experimentally infected animals. Analyses of tissue samples with this probe show that the virus infects connective tissues of gills and hepatopancreas. This is the first gene probe to be constructed for a native viral pathogen of temperate water brachyurans and it will be useful to study virus ecology and virus-host interactions in vivo and in vitro. An understanding of these processes is essential to control and manage disease and, ultimately, to identify immune effectors capable of destroying viral pathogens.