First transcriptome profiling in gill and hepatopancrease tissues of Metapenaeus ensis in response to acute ammonia-N stress.

The greasyback shrimp, Metapenaeus ensis, suffers from ammonia-N stress during intensive factory aquaculture. Optimizing ammonia-N stress tolerance has become an important issue in M. ensis breeding. The metabolic and adaptive mechanisms of ammonia-N toxicity in M. ensis have not been comprehensively understood yet. In this study, a large number of potential simple sequence repeats (SSRs) in the transcriptome of M. ensis were identified. Differentially expressed genes (DEGs) in the gill and hepatopancreas at 24 h post-challenges under high concentrations of ammonia-N treatment were detected. We obtained 20,108,851-27,681,918 clean reads from the control and high groups, assembled and clustered a total of 103,174 unigenes with an average of 876 bp and an N50 of 1189 bp. Comparative transcriptome analyses identified 2000 different expressed genes in the gill and 2010 different expressed genes in the hepatopancreas, a large number of which were related to immune function, oxidative stress, metabolic regulation, and apoptosis. The results suggest that M. ensis may counteract ammonia-N toxicity at the transcriptome level by increasing the expression of genes related to immune stress and detoxification metabolism, and that selected genes may serve as molecular indicators of ammonia-N. By exploring the genetic basis of M. ensis' ammonia-N stress adaptation, we constructed the genetic networks for ammonia-N adaptation. These findings will accelerate the understanding of M. ensis' ammonia-N adaptation, contribute to the research of future breeding, and promote the level of factory aquaculture of M. ensis.

Comparative transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

Transcriptome analysis of hepatopancreas in penaeus monodon under acute low pH stress.

The decrease of seawater pH can affect the metabolism, acid-base balance, immune response and immunoprotease activity of aquatic animals, leading to aquatic animal stress, impairing the immune system of aquatic animals and weakening disease resistance, etc. In this study, we performed high-throughput sequencing analysis of the hepatopancreas transcriptome library of low pH stress penaeus monodon, and after sequencing quality control, a total of 43488612-56271828 Clean Reads were obtained, and GO annotation and KEGG pathway enrichment analysis were performed on the obtained Clean Reads, and a total of 395 DEGs were identified. we mined 10 differentially expressed and found that they were significantly enriched in the Metabolic pathways (ko01100), Biosynthesis of secondary metabolites (ko01110), Nitrogen metabolism (ko00910) pathways, such as PIGA, DGAT1, DGAT2, UBE2E on Metabolic pathways; UGT, GLT1, TIM genes on Biosynthesis of secondary metabolites; CA, CA2, CA4 genes on Nitrogen metabolism, are involved in lipid metabolism, induction of oxidative stress and inflammation in the muscular body of spot prawns. These genes play an important role in lipid metabolism, induction of oxidative stress and inflammatory response in the muscle of the shrimp. In summary, these genes provide valuable reference information for future breeding of low pH-tolerant shrimp.

Identification of multifunctionality of the PmE74 gene and development of SNPs associated with low salt tolerance in Penaeus monodon.

Members of the E74-like factor (ELF) subfamily are involved in the immune stress process of organisms by regulating immune responses and the development of immune-related cells. PmE74 of Penaeus monodon was characterized and functionally analyzed in this study. The full length of PmE74 was 3106 bp, with a 5'-UTR of 297 bp, and a 3'-UTR of 460 bp. The ORF (Open reading frame) was 2349 bp and encoded 782 amino acids. Domain analysis showed that PmE74 contains a typical Ets domain. Multiple sequence alignment and phylogenetic tree analysis showed that PmE74 clustered with Litopenaeus vannamei E74 and displayed significant similarity (98.98%). PmE74 was expressed in all tissues tested in P. monodon, with the highest levels of expression observed in the testis, intestine, and epidermis. Different pathogen stimulation studies have revealed that PmE74 expression varies in response to different pathogen stimuli. A 96-h acute low salt stress study revealed that PmE74 in the hepatopancreas was upregulated and downregulated in the salinity 17 group and considerably downregulated in the salinity 3 group, whereas PmE74 in gill tissue was considerably downregulated in both groups. Further, by knocking down PmE74 and learning the trends of its linkage genes PmAQP1, PmNKA, PmE75, PmFtz-f1, PmEcR, and PmRXR in response to low salt stress, it was further indicated that PmE74 could have a vital role in the regulation of low salt stress. The SNP test revealed that PmE74-In1-53 was significantly associated with low salt tolerance traits in P. monodon (P < 0.05). The findings of this study can aid in the advancement of molecular marker-assisted breeding in P. monodon, as well as provide fundamental data and methodologies for further investigation of its low salt tolerance strains in P. monodon.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-ß-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.

Transcriptome and molecular regulatory mechanisms analysis of gills in the black tiger shrimp Penaeus monodon under chronic low-salinity stress.

Background: Salinity is one of the main influencing factors in the culture environment and is extremely important for the survival, growth, development and reproduction of aquatic animals. Methods: In this study, a comparative transcriptome analysis (maintained for 45 days in three different salinities, 30 psu (HC group), 18 psu (MC group) and 3 psu (LC group)) was performed by high-throughput sequencing of economically cultured Penaeus monodon. P. monodon gill tissues from each treatment were collected for RNA-seq analysis to identify potential genes and pathways in response to low salinity stress. Results: A total of 64,475 unigenes were annotated in this study. There were 1,140 upregulated genes and 1,531 downregulated genes observed in the LC vs. HC group and 1,000 upregulated genes and 1,062 downregulated genes observed in the MC vs. HC group. In the LC vs. HC group, 583 DEGs significantly mapped to 37 signaling pathways, such as the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway; in the MC vs. HC group, 444 DEGs significantly mapped to 28 signaling pathways, such as the MAPK signaling pathway, Hippo signaling pathway and calcium signaling pathway. These pathways were significantly associated mainly with signal transduction, immunity and metabolism. Conclusions: These results suggest that low salinity stress may affect regulatory mechanisms such as metabolism, immunity, and signal transduction in addition to osmolarity in P. monodon. The greater the difference in salinity, the more significant the difference in genes. This study provides some guidance for understanding the low-salt domestication culture of P. monodon.

Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway.

Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH(4)Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase ß.

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase ß protein (scIKKß), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKß. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

Transcriptome reveals the important role of metabolic imbalances, immune disorders and apoptosis in the treatment of Procambarus clarkii at super high temperature.

Temperature is an important environmental factor in the living environment of crustaceans. Changes in temperature can affect their normal growth and metabolism and even cause bacterial disease. Currently, the potential anti-reverse molecular reaction mechanism of crustaceans during high-temperature conditions has not yet been fully understood. Therefore, in this study, we characterised the transcriptome of Procambarus clarkii using RNA sequencing and performed a comparison between super-high-temperature treated samples and controls. After assembly and annotation, 81,097 unigenes with an average length of 069 bp and 358 differentially expressed genes (DEGs) were identified. Among these DEGs, 264 were differentially upregulated and 94 were differentially downregulated. To obtain comprehensive gene function information, we queried seven databases, namely, Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO to annotate gene functions. Transcriptome analysis revealed that the identified DEGs have significant effects on immune-related pathways, including lysosomal and phagosomal pathways, and that super-high-temperature conditions can cause disease in P. clarkii. Some significantly downregulated genes are involved in oxidative phosphorylation and the PPAR signalling pathway; this suggests a metabolic imbalance in P. clarkia during extreme temperature conditions. In addition, elevated temperature changed the expression patterns of key apoptosis genes XIAP, CASP2, CASP2, CASP8, and CYTC, thereby confirming that high-temperature conditions caused immune disorders, metabolic imbalance, and, finally, triggered apoptosis. Our results provide a useful foundation for understanding the molecular mechanisms underlying the responses of P. clarkii during high-temperature conditions.

The JAK and STAT family members of the mandarin fish Siniperca chuatsi: molecular cloning, tissues distribution and immunobiological activity.

The JAK/STAT signal transduction pathway plays a critical role in host defence against viral and bacterial infections. In the present study, we report cDNA cloning and characterization of the JAK family (mJAK1-3 and mTYK2) and STAT family members (mSTAT1, mSTAT3-6) from the mandarin fish Siniperca chuatsi. To our knowledge, JAK2, TYK2 and STAT6 genes were cloned from fish for the first time. The mJAK family proteins consist of 1112-1177 residues with a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. The mSTAT family members contain 716-786 residues with similar architecture, including an N-terminal domain, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcription activation domain. Multiple sequence alignments of mJAKs/mSTATs and phylogenetic analysis showed that mJAK1 was closed to mTYK2, and mJAK2 was closed to mJAK3. Quantitative real-time PCR results revealed that mJAK/mSTAT family members were expressed in most tissues examined except muscle. In mandarin fish fry cells, the expressions of IRF-1, Mx, SOCS1 and SOCS3 genes were significantly induced by poly(I:C) stimulation, indicating that the mJAK/mSTAT signal pathway is activated by poly(I:C). Furthermore, expressions of all four mJAKs and four mSTATs were all up-regulated after poly(I:C) stimulation, but expression of mSTAT5 was inhibited by poly(I:C). These results suggest that mandarin fish has the JAK/STAT signal transduction pathways similar to those in mammals, and these signalling pathways may play an important role in regulation of antiviral responses in fish.

Identification and functional study of a shrimp Relish homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here we describe the identification and functional analysis of a Relish homologue, LvRelish and its shorter isoform sLvRelish, from the Pacific white shrimp, Litopenaeus vannamei. The LvRelish gene has 22 exons in approximately 15 kb genomic sequence. The full-length cDNA of LvRelish is 4071 bp with an open reading frame that encodes 1207 amino acids. LvRelish contains a conserved Rel homology domain (RHD), a nucleus localization signal, an IkappaB-like domain (six ankyrin repeats), and a death domain, suggesting that it belongs to the class I NF-kappaB. sLvRelish cDNA is 1051 bp encoding 317 amino acids. It shares the RHD region with LvRelish. RT-PCR analysis showed that LvRelish and sLvRelish mRNAs were expressed at different levels in tissues. Western blot analysis showed that recombinant intact LvRelish could be cleaved into two fragments in S2 cells, and immunofluorescence assay showed that the plasmid-expressed LvRelish protein was seen both in the cytoplasm and the nucleus. Electrophoretic mobility shift assay showed that recombinant RHD of LvRelish in S2 cells bound specifically with Drosophila melanogaster kappaB motifs in vitro. Both the LvRelish and its RHD domain transactivated the reporter gene controlled by the 5' flanking region of penaeidin 4, an antibacterial peptide of shrimp, suggesting that LvRelish can regulate the transcription of penaeidin 4 gene. Identification of LvRelish will help us better understand shrimp immunity and may help obtain more effective methods to prevent shrimp diseases.

A Toll receptor in shrimp.

Outbreaks of infectious diseases have resulted in high mortality and huge economic losses in penaeid shrimp culture. Interest in understanding shrimp immunity has increased because of its importance in disease control. Here we report cDNA cloning of a Toll receptor from the white shrimp Litopenaeus vannamei. L. vannamei Toll (lToll) is 926 residues, with a putative signal peptide of 19 residues. The protein contains distinct structural/functional motifs of the Toll like receptor (TLR) family, including an extracellular domain containing 16 leucine-rich repeats (LRRs) flanked by cysteine-rich motifs and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. The lToll TIR domain showed high similarity to Apis mellifer Toll and Drosophila melanogaster Toll, with 59.9% and 54.3% identity, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that lToll was expressed in hemocyte, gill, heart, brain, stomach, intestine, pyloric caecum, muscle, nerve and spermary, with a lower expression level in eyestalk and hepatopancreas. Identification of lToll will help to elucidate the Toll pathway in shrimp innate immunity.

[Effects of different doses and concentrations of ropivacaine in axillary brachial plexus block].

OBJECTIVE: To evaluate clinic pharmacodynamics of ropivacaine hydrochloride, a new long-acting amide type local anaesthetic, in Chinese patients undergoing neurolysis and tendolysis of forearm through axillary brachial plexus block. METHODS: Two hundred ASAI-II persons, aged 18-65,weighing 60-75 kg, undergoing neurolysis and tendolysis of forearm under axillary brachial plexus block were randomly assigned to 4 equal groups to received ropivacaine of the doses of 1.00 mg/kg (Group I), 1.25 mg/kg (Group II), 1.50 mg/kg (Group III), and 2.00 mg/kg (Group IV). Each group was subdivided into 5 equal subgroups according to the concentrations (0.15%, 0.20%, 0.25%, 0.30%, and 0.35%). The effects were analyzed. Results There was no significant difference in starting time, consummating time, and persisting time of sensory and motor nerve block between Group I and Group II (all P > 0.05). The starting time and consummating time of Group III and Group IV were all significantly shorter than those of Group I (all P < 0.05), and the persisting times of Groups III and IV were all significantly longer than those of Group I (P < 0.05). 90 minutes after brachial plexus block the degrees of muscle strength Groups I and II were M1 and M2 respectively, hence, the sensory and motor nerves block was in dissociation. When motor never block was above M3 and M4 the interval of brachial plexus block was 55 min and 27 min in Group III and Group IV respectively. CONCLUSION: Sensory and motor never can be blocked perfectly when the dose of ropivacaine is between 1.5 mg/kg and 2.0 mg/kg and the concentration is between 0.30%-0.35%. When the doses of ropivacaine is 1.25 mg/kg and the concentration is 0.20%-0.25% the starting and consummating time are longer and persisting time is shorter. When the doses of ropivacaine is 1.00 mg/kg and the concentration is 0.15%-0.20% the starting and consummating time are longer and only satisfies external debridement and suture without tourniquets.

Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA.

RNA interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation. Here, we described specific silencing of five white spot syndrome virus (WSSV) genes in Litopenaeus vannamei in vivo with sequence-specific siRNAs. These genes included DNA polymerase (dnapol), ribonucleotide reductase small subunit (rr2), thymidine kinase and thymidylate kinase (tk-tmk), vp24 and vp28. At 6 days post-challenged, the relative survival rates of shrimp injected with siDNApol, siRR2, siTK-TMK, siVP24 and siVP28 (siRNAs for dnapol, rr2, tk-tmk, vp24 and vp28 genes) reached 50%, 50%, 66%, 33% and 33%, respectively. Specific siRNAs of the five WSSV genes could result in suppression of the target genes and a significant reduction in the viral proliferation. In negative controls, sequence-independent siRNA (mutant siRNA) could not inhibit expression of these five genes or viral replication. Consequently, injection of sequence-dependent siRNA could induce anti-WSSV activity in shrimp. These results suggest that siRNA can suppress WSSV efficiently in shrimp, and it may provide a potential approach to the therapy of aquaculture viral disease.

Nucleic acid-induced antiviral immunity in shrimp.

Vertebrates detect viral infection predominantly by sensing viral nucleic acids to produce type I interferon (IFN). In invertebrates, it has been believed that the IFN system is absent and RNA interference is a sequence-specific antiviral pathway. In this study, we found that injection of nucleic acid mimics poly(I:C), poly(C:G), CL097, poly C and CpG-DNA, afforded shrimp antiviral immunity, which is similar to the vertebrate IFN system. Using suppression subtractive hybridization (SSH) method, 480 expression sequence tags were identified to be involved in the poly(I:C)-induced antiviral immunity of the model crustacean Litopenaeus vannamei, and 41% of them were new genes. In the SSH libraries, several IFN system-related genes such as dsRNA-dependent protein kinase PKR, Toll-like receptor 3 (TLR3) and IFNγ-inducible protein 30 were identified. L. vannamei IKKε, whose vertebrate homologs are central regulators of the IFN-producing pathway, could significantly activate IFN reporter genes in HEK293T cells. In crustacean databases, many genes homologous to genes of the vertebrate IFN response, such as IRFs, PKR, ADAR (adenosine deaminase, RNA-specific) and other interferon-stimulated genes (ISGs) were discovered. These results suggest that shrimp may possess nucleic acid-induced antiviral immunity.

A novel viral SOCS from infectious spleen and kidney necrosis virus: interacts with Jak1 and inhibits IFN-α induced Stat1/3 activation.

Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.

Involvement of caveolin-1 in the Jak-Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi).

Caveolae, the major source of caveolin-1 protein, are specialized invaginated microdomains of the plasma membrane that act as organizing centers for signaling molecules in the immune system. In the present study, we report the cloning and characterization of caveolin-1 (mCav-1) from mandarin fish (Siniperca chuatsi) and study on the roles of mCav-1 in the fish Jak-Stat signaling pathway and in virus infection. The cDNA sequence of mCav-1 was 707bp in size, encoding a protein of 181 amino acids, which was different from the mammalian protein (178 amino acids). The deduced amino acid sequence of mCav-1 shared similar architecture with vertebrate caveolin-1 proteins, but mCav-1 lacked a phosphorylation site (y14). The major subcellular location of mCav-1 was in the caveolae, where the protein appeared to have major functions. Real-time PCR revealed that the expression of the mandarin fish Mx, IRF-1, SOCS1, and SOCS3 genes involved in the poly(I:C)-induced Jak-Stat signaling pathway was impaired by the mCav-1 scaffolding domain peptide (mSDP). In mandarin fish fry (MFF-1) cells, the protein levels of mCav-1 were markedly up-regulated at 12 and 24h post-infection with ISKNV (infectious spleen and kidney necrosis virus). In addition, ISKNV entry into MFF-1 cells was significantly inhibited by mSDP, and the inhibition was dose-dependent. Thus, ISKNV infection was apparently associated with mCav-1 protein and may utilize the caveolae-related endocytosis pathway. The findings reported here further our understanding of the function of caveolin-1 in the complex signal transduction network in fish immune systems and in the cellular entry mechanism of iridoviruses.

Identification and functional study of a shrimp Dorsal homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here, identification and functional analysis of LvDorsal, a Dorsal homologue from the Pacific white shrimp Litopenaeus vannamei, were described. The full-length cDNA of LvDorsal is 2204bp with an open reading frame that encodes 400 amino acids. The deduced LvDorsal contains a conserved Rel homology domain (RHD), an IPT (Ig-like, plexins and transcription factors) domain and a nucleus localization signal, suggesting that it belongs to the class II NF-kappaB. RT-PCR analysis showed that LvDorsal mRNAs were expressed in all the tissues tested, including gill, epidermis, hemocytes, intestine, stomach, eyestalk, brain, hepatopancreas, muscle, heart and pyloric caecum. Immunofluorescence assay showed that recombinant LvDorsal was translocated into the nucleus of Drosophila S2 cells. Electrophoretic mobility shift assay illustrated that recombinant LvDorsal RHD from S2 cells bound specifically with D. melanogaster kappaB motifs. Additionally, the dual-luciferase reporter assays indicated that LvDorsal could transactivate the reporter gene controlled by the 5' flanking region of shrimp penaeidin-4 and Drosophila attacin genes, suggesting that LvDorsal can regulate the transcription of shrimp penaeidin-4 gene. Study of LvDorsal will help us to better understand shrimp immunity and may help to obtain more effective methods to prevent shrimp diseases.