Molecular mechanism of the interaction between Megalocytivirus-induced virus-mock basement membrane (VMBM) and lymphatic endothelial cells.

IMPORTANCE: Viruses are able to mimic the physiological or pathological mechanism of the host to favor their infection and replication. Virus-mock basement membrane (VMBM) is a Megalocytivirus-induced extracellular structure formed on the surface of infected cells and structurally and functionally mimics the basement membrane of the host. VMBM provides specific support for lymphatic endothelial cells (LECs) rather than blood endothelial cells to adhere to the surface of infected cells, which constitutes a unique phenomenon of Megalocytivirus infection. Here, the structure of VMBM and the interactions between VMBM components and LECs have been analyzed at the molecular level. The regulatory effect of VMBM components on the proliferation and migration of LECs has also been explored. This study helps to understand the mechanism of LEC-specific attachment to VMBM and to address the issue of where the LECs come from in the context of Megalocytivirus infection.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei.

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.

Honokiol induces apoptosis-like death in Cryptocaryon irritans Tomont.

BACKGROUND: Cryptocaryon irritans, a common parasite in tropical and subtropical marine teleost fish, has caused serious harm to the marine aquaculture industry. Honokiol was proven to induce C. irritans tomont cytoplasm shrinkage and death in our previous study, but the mechanism by which it works remains unknown. METHODS: In this study, the changes of apoptotic morphology and apoptotic ratio were detected by microscopic observation and AnnexinV-FITC/PI staining. The effects of honokiol on intracellular calcium ([Ca2+]i) concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF) and caspase activities were detected by Fluo-3 staining, JC-1 staining, DCFH-DA staining, Tunel method and caspase activity assay kit. The effects of honokiol on mRNA expression levels of 61 apoptosis-related genes in tomonts of C. irritans were detected by real-time PCR. RESULTS: The results of the study on the effects of honokiol concentration on C. irritans tomont apoptosis-like death showed that the highest levels of prophase apoptosis-like death rate (PADR), [Ca2+]i concentration, ROS, the activities of caspase-3/9 and the lowest necrosis ratio (NER) were obtained at a concentration of 1 µg/ml, which was considered the most suitable for inducing C. irritans tomont apoptosis-like death. When C. irritans tomonts were treated with 1 µg/ml honokiol, the [Ca2+]i concentration began to increase significantly at 1 h. Following this, the ROS, QDF and activities of caspase-3/9 began to increase significantly, and the ΔΨm began to decrease significantly at 2 h; the highest PADR was obtained at 4 h. The mRNA expression of 14 genes was significantly upregulated during honokiol treatment. Of these genes, itpr2, capn1, mc, actg1, actb, parp2, traf2 and fos were enriched in the pathway related to apoptosis induced by endoplasmic reticulum (ER) stress. CONCLUSIONS: This article shows that honokiol can induce C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca2+]i homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway, which might represent a novel therapeutic intervention for C. irritans infection.

Production and characterization of monoclonal antibodies against mandarinfish ranavirus and first identification of pyloric caecum as the major target tissue.

Mandarinfish ranavirus (MRV), also known as a variant of largemouth bass virus (LMBV), is an emerging pathogen in mandarinfish aquaculture. In this study, monoclonal antibodies (mAbs) against MRV were produced and characterized, and 7 mAbs were obtained through Western blotting screening and all 7 mAbs specifically recognized MRV/LMBV but not several piscine iridoviruses as ISKNV, GIV and TFV. By LC MS/MS analysis, the recognized viral proteins by seven mAbs were identified as MRV-pORF47L, MRV-pORF55R, MRV-pORF57L, MRV-pORF77L and MRV-pORF78L, respectively, and all five viral proteins are late expression structural proteins by Western blotting. Based on mAb 1C4, immuno-histochemistry and immuno-histo-fluorescence were performed to re-assess the tissue tropism of MRV. The result showed that abundant reactive signals were observed in infected spleen, kidney as well as intestine and pyloric caecum. Real-time quantitative PCR also demonstrated that spleen as well as pyloric caecum and intestines are the major target tissue upon MRV infection. In infected intestines and pyloric caecum, numerous enlarged, multinucleated cells with intracytoplasmic inclusions were identified as the target cells of MRV, suggesting that MRV serves as a digestive tract pathogen to mandarinfish, which may explain why acute infection of MRV can cause the typical clinicopathology featured by severe ascites.

White Spot Syndrome Virus (WSSV) Inhibits Hippo Signaling and Activates Yki To Promote Its Infection in Penaeus vannamei.

White spot syndrome virus (WSSV) is a serious threat to shrimp aquaculture, especially Pacific white shrimp, Penaeus vannamei, the most farmed shrimp in the world. Activation of the Hippo-Yki signaling pathway, characterized by the intracellular Hippo-Wts kinase cascade reactions and the phosphorylation and cytoplasmic retention of Yki, is widely involved in various life activities. The current work established the fundamental structure and signal transduction profile of the Hippo-Yki pathway in P. vannamei and further investigated its role in viral infection. We demonstrated that WSSV promoted the dephosphorylation and nuclear translocation of Yki, suggesting that Hippo signaling is impaired and Yki is activated after WSSV infection in shrimp. In vivo, Yki gene silencing suppressed WSSV infection, while Hippo and Wts silencing promoted it, indicating a positive role of Hippo signaling in antiviral response. Further analyses showed that Yki suppressed Dorsal pathway activation and inhibited hemocyte apoptosis in WSSV-infected shrimp, while Hippo and Wts showed opposite effects, which contributed to the role of Hippo signaling in WSSV infection. Therefore, the current study suggests that WSSV annexes Yki to favor its infection in shrimp by inhibiting Hippo signaling. IMPORTANCE White spot syndrome virus (WSSV) is one of the most harmful viral pathogens to shrimp. The pathological mechanism of WSSV infection remains unclear to date. The Hippo-Yki signaling pathway is important for various biological processes and is extensively involved in mammalian immunity, but little is known about its role in infectious diseases in invertebrates. Based on revealing the fundamental structure of the shrimp Hippo pathway, this study investigated its implication in the pathogenesis of WSSV disease. We demonstrated that WSSV enhanced Yki activation by inhibiting Hippo signaling in shrimp. The activated Yki promoted WSSV infection by inhibiting hemocyte apoptosis and suppressing the activation of Dorsal, an NF-κB family member in shrimp that is critical for regulating antiviral response. Therefore, this study suggests that WSSV can hijack the Hippo-Yki signaling pathway to favor its infection in shrimp.

Virome Analysis of Normal and Growth Retardation Disease-Affected Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species in China. Growth retardation disease (GRD) is a common contagious disease in M. rosenbergii, resulting in slow growth and precocious puberty in prawns, and has caused growing economic losses in the M. rosenbergii industry. To investigate the viral diversity of M. rosenbergii and identify potentially high-risk viruses linked to GRD, virome analysis of the GRD-affected and normal M. rosenbergii was carried out using next-generation sequencing (NGS). A total of 327 contigs (>500 bp) were related to viral sequences belonging to 23 families/orders and a group of unclassified viruses. The majority of the viral contigs in M. rosenbergii belonged to the order Picornavirales, with the Solinviviridae family being the most abundant in both the diseased and normal groups. Furthermore, 16 RNA viral sequences with nearly complete genomes were characterized and phylogenetically analyzed, belonging to the families Solinviviridae, Flaviviridae, Polycipiviridae, Marnaviridae, and Dicistroviridae as well as three new clades of the order Picornavirales. Notably, the cross-species transmission of a picorna-like virus was observed between M. rosenbergii and plants. The "core virome" seemed to be present in the diseased and normal prawns. Still, a clear difference in viral abundance was observed between the two groups. These results showed that the broad diversity of viruses is present in M. rosenbergii and that the association between viruses and disease of M. rosenbergii needs to be further investigated. IMPORTANCE Growth retardation disease (GRD) has seriously affected the development and economic growth of the M. rosenbergii aquaculture industry. Our virome analysis showed that diverse viral sequences were present in M. rosenbergii, significantly expanding our knowledge of viral diversity in M. rosenbergii. Some differences in viral composition were noted between the diseased and normal prawns, indicating that some viruses become more abundant in occurrences or outbreaks of diseases. In the future, more research will be needed to determine which viruses pose a risk for M. rosenbergii. Our study provides important baseline information contributing to disease surveillance and risk assessment in M. rosenbergii aquaculture.

Neomycin inhibits Megalocytivirus infection in fish by antagonizing the increase of intracellular reduced glutathione.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus that infects a number of marine and freshwater fishes, causing huge economic losses in aquaculture. The ISKNV infection leads to increase of reducing power in cells. As the antibiotic neomycin can promote the production of reactive oxygen species (ROS) in animal cells, in the current study, the potential therapeutic effect of neomycin on ISKNV infection was explored. We showed that neomycin could decrease the reducing power in cultured MFF-1 cells and inhibit ISKNV infection by antagonizing the shift of the cellular redox balance toward reduction. In vivo experiments further demonstrated that neomycin treatment significantly suppresses ISKNV infection in mandarin fish. Expression of the major capsid protein (MCP) and the proportion of infected cells in tissues were down-regulated after neomycin treatment. Furthermore, neomycin showed complex effects on expression of a set of antiviral related genes of the host. Taking together, the current study suggested that the viral-induced redox imbalance in the infected cells could be used as a target for suppressing ISKNV infection. Neomycin can be potentially utilized for therapeutic treatment of Megalocytivirus diseases by antagonizing intracellular redox changes.

Insights into the synergy between functional microbes and dissolved oxygen partition in the single-stage partial nitritation-anammox granules system.

The rapid start-up and stable operation of the single-stage partial nitritation-anammox (PNA) process remains a challenge in practical applications. An integrated investigation of nitrogen removal performance, sludge characteristics, activity and abundance, and microbial dynamics was implemented for 360 days via an airlift internal circulation reactor. During long-term operation, the reactor realized a stable dissolved oxygen (DO) partition and cultivated granular sludge. The nitrogen removal rate increased from 0.15 kg-N/m3/d to 1.24 kg-N/m3/d, and a high nitrogen removal efficiency of 82.6% was obtained. A stable DO partition further accelerated the bioreaction rates and enhanced the activity of functional microbes. The activities of ammonia oxidation and anammox reached 1.21 g-N/g-VSS/d and 1.43 g-N/g-VSS/d, respectively. Sludge granulation efficiently enriched the abundances of Candidatus Brocadia (7.4%) and Nitrosomonas (5.2%). These results demonstrated that efficient DO partition and stable culture of granular sludge could enhance the synergy of functional microbes for autotrophic nitrogen removal.

A Dicer2 from Scylla paramamosain activates JAK/STAT signaling pathway to restrain mud crab reovirus.

A Dicer2 gene from Scylla paramamosain, named SpDicer2, was cloned and characterized. The full length of SpDicer2 mRNA contains a 121 bp 5'untranslated region (UTR), an open reading frame (ORF) of 4518 bp and a 3' UTR of 850 bp. The SpDicer2 protein contains seven characteristic Dicer domains and showed 34%-65% identity and 54%-79% similarity to other Dicer protein domains, respectively. The mRNA of SpDicer2 was high expressed in hemocytes, intestine and gill and low expressed in the eyestalk and muscle. Moreover, expression of SpDicer2 was significantly responsive to challenges by mud crab reovirus (MCRV), Poly(I:C), LPS, Staphylococcus aureus and Vibrio parahaemolyticus. SpDicer2 was dispersedly presented in the cytoplasm except for a small amount in the nucleus. SpDicer2 could activate SpSTAT to translocate from the cytoplasm to the nucleus, and significantly increase the transcription activity of the wsv069 promoter, suggesting that SpDicer2 activated the JAK/STAT pathway. Furthermore, silencing of SpDicer2 in vivo increased the mortality of MCRV infected mud crab and the viral load in tissues and down-regulated the expression of multiple components of Toll, IMD and JAK-STAT pathways and almost all the examined immune effector genes. These results suggested that SpDicer2 could play an important role in defense against MCRV via activating the JAK/STAT signaling pathways in mud crab.

A Novel Forkhead Box Protein P (FoxP) From Litopenaeus vannamei Plays a Positive Role in Immune Response.

The forkhead box protein P (FoxP) family members have been known to be important for regulation of immune responses in vertebrates, but their roles in invertebrate immunity remain unclear. In this study, a novel FoxP gene (LvFoxP) was identified from Pacific white shrimp Litopenaeus vannamei and functionally studied in the context of immune response. Possessing a conserved FoxP coiled-coil domain and a forkhead domain, LvFoxP shared homology to vertebrate FoxP family members, in particular FoxP1. Expression of LvFoxP was detectable in all the examined tissues and could be up-regulated by immune challenge in gill and hemocytes. The LvFoxP protein was present in both the cytoplasm and nucleus of hemocytes and could be nuclear-translocated upon immune stimulation. Silencing of LvFoxP increased the susceptibility of shrimp to infections by Vibrio parahaemolyticus and white spot syndrome virus (WSSV) and down-regulated the expression of multiple components of NF-κB and JAK-STAT pathways and almost all the examined immune effector genes. Moreover, the phagocytic activity of hemocytes from LvFoxP-silenced shrimp against V. parahaemolyticus was decreased. These suggested that LvFoxP could play a positive role in immune response. The current study may provide novel insights into the immunity of invertebrates and the functional evolution of the FoxP family.

The NF-κB family member dorsal plays a role in immune response against Gram-positive bacterial infection in mud crab (Scylla paramamosain).

The NF-κB family is a set of evolutionarily conserved transcription factors that play central roles in various biological events. Dorsal is an invertebrate NF-κB family member that is essential for the regulation of immune responses. In the current study, the Dorsal gene from Scylla paramamosain (SpDorsal) was identified, which showed high homology to other crustacean Dorsal proteins. Expression of SpDorsal was highest in hemocytes and could be significantly changed after immune stimulations. In expression vector-transfected S2 cells, SpDorsal was mainly localized in the cytoplasm and could be efficiently translocated into the nucleus upon immune stimulations with the Gram-positive bacteria Staphylococcus aureus and poly (I:C), but not the Gram-negative bacteria Vibrio parahaemolyticus. As a transcription factor, SpDorsal could activate the promoter of S. paramamosain Hyastatin (SpHyastatin) in vitro, while S. paramamosain Cactus (SpCactus), a homolog of IκB, could interact with SpDorsal to prevent its nuclear translocation and inhibit its transcription factor activity. Silencing of SpDorsal in vivo using RNAi strategy significantly increased the mortality of crabs infected with S. aureus but not that with V. parahaemolyticus. These indicated that the SpDorsal signaling pathway could be mainly implicated in immune responses against Gram-positive bacterial infection in S. paramamosain.

A signal transducers and activators of transcription (STAT) gene from Scylla paramamosain is involved in resistance against mud crab reovirus.

A STAT gene from Scylla paramamosain, named SpSTAT, was cloned and characterized. The full length of SpSTAT mRNA contains a 5'untranslated region (UTR) of 238 bp, an open reading frame (ORF) of 2388 bp and a 3' UTR of 326 bp. The SpSTAT protein contains four characteristic STAT domains and showed 84% identity (90% similarity) and 44% identity (64% similarity) to Litopenaeus vannamei STAT protein and Human STAT5a/b protein, respectively. The mRNA of SpSTAT was high expressed in the intestine and eyestalk and low expressed in the heart and muscle. Moreover, expression of SpSTAT was significantly responsive to challenge of mud crab reovirus (MCRV), Poly(I:C), LPS and Staphylococcus aureus. SpSTAT could be activated by Poly(I:C) and LPS to translocate to the nucleus of Drosophila Schneider 2 (S2) cells. SpSTAT could be phosphorylated by interaction with JAK of S. paramamosain (SpJAK) and activated to translocate to the nucleus of S2 cells. Furthermore, Silencing of SpSTAT in vivo resulted in higher mortality rate of MCRV infected mud crab and increased the viral load in tissues, suggesting that SpSTAT could play an important role in defense against MCRV in mud crab.

A janus kinase from Scylla paramamosain activates JAK/STAT signaling pathway to restrain mud crab reovirus.

JAK/STAT signaling pathways are associated with the innate immune system and play important roles in mediating immune responses to virus infection. In this study, a Janus kinase gene from Scylla paramamosain (SpJAK) was cloned and characterized. The full length of SpJAK mRNA contains a 5' untranslated region (UTR) of 304 bp, an open reading frame of 3300 bp and a 3' UTR of 302 bp. The SpJAK protein contains seven characteristic JAK homology domains (JH1 to JH7) and showed 60% identity (78% similarity), 20% identity (35% similarity), and 21% identity (37% similarity) to the Litopenaeus vannamei JAK (LvJAK) protein, the Drosophila melanogaster hopscotch protein, and the Homo sapiens JAK2 protein, respectively. The mRNA of SpJAK showed high expression in the brain and nerve but low expression in the hemocyte and muscle. Moreover, the expression of SpJAK was significantly upregulated by stimulation with mud crab reovirus (MCRV), poly(I:C), and Vibrio parahaemolyticus. SpJAK significantly activated the STAT of S. paramamosain (SpSTAT) to translocate to the nucleus of Drosophila Schneider 2 cells. SpJAK significantly enhanced the activity of the promoter of the WSSV wsv069 gene that was activated significantly by SpSTAT by acting on the STAT-binding DNA motif. These results suggest that SpJAK activates the JAK/STAT pathway. Furthermore, silencing SpJAK in vivo resulted in the high mortality rate of MCRV-infected mud crabs and increased the viral load in tissues. Hence, SpJAK could play an important role in defense against MCRV in mud crab.

Fatty acid synthase plays a positive role in shrimp immune responses against Vibrio parahaemolyticus infection.

Fatty acid synthase (FAS) is an important enzyme that catalyzes the synthesis of fatty acids. In this study, the role of the FAS gene from pacific white shrimp Litopenaeus vannamei (LvFAS) in immune responses against Vibrio parahaemolyticus infection was studied. The expression of LvFAS could be up-regulated upon infection of V. parahaemolyticus and stimulation of lipopolysaccharide and poly (I:C). The promoter of LvFAS was predicted to harbor a NF-κB binding site and dual-luciferase reporter assays demonstrated that the NF-κB family proteins Relish, sRelish and Dorsal could activate the transcription of LvFAS. After knockdown of LvFAS expression using RNAi strategy, both the mortality of V. parahaemolyticus infected shrimps and the bacterial load in shrimp tissues were significantly increased. Meanwhile, the expression of many immune-responsive genes, such as antimicrobial peptides, C-type lectins (CTLs), lysozyme and hemolin, was down-regulated. These suggested that LvFAS could play a positive role in anti-V. parahaemolyticus responses in shrimp. To our knowledge, this is the first study that investigates the role of FAS in antibacterial immunity in animals, which may indicate the relationship between the anabolism of fatty acids and immune responses in crustaceans.

Transcriptome analysis of mud crab (Scylla paramamosain) gills in response to Mud crab reovirus (MCRV).

Mud crab (Scylla paramamosain) is an economically important marine cultured species in China's coastal area. Mud crab reovirus (MCRV) is the most important pathogen of mud crab, resulting in large economic losses in crab farming. In this paper, next-generation sequencing technology and bioinformatics analysis are used to study transcriptome differences between MCRV-infected mud crab and normal control. A total of 104.3 million clean reads were obtained, including 52.7 million and 51.6 million clean reads from MCRV-infected (CA) and controlled (HA) mud crabs respectively. 81,901, 70,059 and 67,279 unigenes were gained respectively from HA reads, CA reads and HA&CA reads. A total of 32,547 unigenes from HA&CA reads called All-Unigenes were matched to at least one database among Nr, Nt, Swiss-prot, COG, GO and KEGG databases. Among these, 13,039, 20,260 and 11,866 unigenes belonged to the 3, 258 and 25 categories of GO, KEGG pathway, and COG databases, respectively. Solexa/Illumina's DGE platform was also used, and about 13,856 differentially expressed genes (DEGs), including 4444 significantly upregulated and 9412 downregulated DEGs were detected in diseased crabs compared with the control. KEGG pathway analysis revealed that DEGs were obviously enriched in the pathways related to different diseases or infections. This transcriptome analysis provided valuable information on gene functions associated with the response to MCRV in mud crab, as well as detail information for identifying novel genes in the absence of the mud crab genome database.