The Small GTPase Rab5c Exerts Bi-Function in Singapore Grouper Iridovirus Infections and Cellular Responses in the Grouper, Epinephelus coioides.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides, and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus, and Homo sapiens, respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses.

To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.

Molecular cloning, characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): serum amyloid A.

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.

Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis.

Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.

Molecular cloning, expression analysis and characterization of peroxiredoxin during WSSV infection in shrimp Fenneropenaeus indicus.

Innate immunity is the first line of defense in shrimps against invading pathogenic microorganisms. Peroxiredoxins (PRX) are the family of antioxidant proteins that play a crucial role in reduction of oxidative stress in host during viral infection. Peroxiredoxin from Fenneropenaeus indicus was identified, cloned and expressed in prokaryotic expression system. The Fi-PRX protein consists of two conserved Cys residues and belongs to typical 2-cys PRX family. The phylogenetic analysis revealed a close evolutionary relatedness of Fi-PRX with the PRX from Drosophila melanogaster PRX1 and distant origin with PRX sequences from other shrimp isolates Fenneropenaeus chinensis, Litopenaeus vannamei and Penaeus japonicus. Fi-PRX transcripts are constitutively expressed in hemocytes and tissues (gills, heart and muscle) and down regulated during 12 h, 24 h and 48 h of WSSV challenged shrimps. Fi-PRX protein levels correlated well with the corresponding levels of Fi-PRX transcripts in hemocytes and tissues of WSSV challenged shrimps. Recombinant Fi-PRX reduces insulin only in the presence of DTT suggesting that the antioxidant function of the protein is thiol dependent. These findings suggest that antioxidant activity of Fi-PRX play a significant role in neutralization of excessive free radicals and ROS generated during viral invasion.

Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38ß) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38ß showed high homologies to other fish p38a MPAK, p38b MAPK and p38ß MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38ß are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38ß were different, and Ec-p38ß was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38ß in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38ß inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38ß played a crucial role in regulating apoptosis and virus replication during iridovirus infection.

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.

Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells.

Stress-activated protein kinases (SAPKs), including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), are usually activated in response to different environmental stimuli, including virus infection. In the present study, the roles of SAPKs during Singapore grouper iridovirus (SGIV) infection were investigated in fish cells. The results showed that increased phosphorylation of JNK1/2 and p38 MAPK occurred during active replication of SGIV in grouper cell cultures. Moreover, downstream effectors (c-Jun, MAPK-activated protein kinase 2, p53, activator protein 1, Myc and nuclear factor of activated T cells) were activated after SGIV infection, suggesting that SGIV replication activated the JNK and p38 MAPK signalling pathways. Notably, using specific inhibitors, it was found that viral gene transcripts, protein expression and viral titres were not affected by inhibition of p38 MAPK but were suppressed significantly by inhibiting JNK1/2 activation. In addition, transcription of grouper immune genes including interferon regulatory factor 1, interleukin-8 and tumour necrosis factor alpha (TNF-α) were regulated by JNK, whilst only TNF-α was regulated by p38 MAPK. It is proposed that the JNK pathway is important for SGIV replication and modulates the inflammatory responses during virus infection.

Molecular cloning of IKKß from the mandarin fish Siniperca chuatsi and its up-regulation in cells by ISKNV infection.

The IκB kinase ß (IKKß) plays crucial roles in regulating activation of nuclear factor-kappa B (NF-κB) in response to proinflammatory factors and microbial and viral infections. Here, we report the cloning of an IKKß cDNA (named SicIKKß) from the mandarin fish Siniperca chuatsi. The full-length cDNA is 4052bp and contains an ORF that encodes a predicted protein of 743-amino acid residues. The deduced amino acid sequence of SicIKKß has the same domain organization as human IKKß, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. Quantitative RT-PCR showed that SicIKKß was ubiquitously expressed in tissues of mandarin fish, and its expression in mandarin fish fry (MFF-1) cells was up-regulated during the course of ISKNV infection.

Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus.

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer, muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.

Requirement for shrimp caspase in apoptosis against virus infection.

Caspases are central effectors in apoptosis. In this investigation, a novel caspase gene (designated as PjCaspase) obtained from the marine shrimp Marsupenaeus japonicus was found to be significantly upregulated in survivors of WSSV-challenged shrimp, suggesting that it might be involved in shrimp antiviral immunity. As revealed by RNAi assays, when the PjCaspase gene was silenced by gene-specific siRNA, the WSSV-induced apoptosis was significantly inhibited. The results showed that the PjCaspase gene was essential in the virus-induced apoptosis of shrimp. Based on the quantitative PCR detection, it was shown that the PjCaspase gene silencing resulted in the increase of virus copies, indicating that apoptosis played a key role in antiviral process of shrimp. As well known, caspase-3 and -8 are crucial caspases in apoptosis. The discovery of PjCaspase in this study would contribute another essential caspase involved in apoptosis against virus infection, which might reveal an ancient mechanism of caspase activation in invertebrate immunity against viruses.

Characterization of a Rab GTPase up-regulated in the shrimp Peneaus japonicus by virus infection.

The molecular mechanisms of the immune system against virus in shrimp are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as PjRab gene) was obtained from Peneaus japonicus shrimp, which exhibited high homology with Rab 6 of other species. The PjRab protein, having GTP-binding activity, contained characteristic signatures of Rab proteins with 6 GTP binding domains and 5 Rab specific domains. However, the PjRab protein exhibited a very different prenylation site (CLLNL) at its C-terminus from most of other Rabs. The PjRab gene was ubiquitously expressed in shrimp tissues. Real-time PCR revealed that the PjRab gene was up-regulated in WSSV-resistant shrimp, suggesting that the PjRab protein might play an important role in shrimp immune response against virus infection. This discovery might contribute better understanding to the molecular events involved in shrimp as well as invertebrate immune responses.

The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression.

Manganese containing superoxide dismutase (SOD) is normally a nuclear-encoded mitochondrial enzyme in eukaryotic organisms; however, a cytoplasmic manganese SOD (cMnSOD) was found in crustaceans that use hemocyanin as oxygen carrier. The complete cDNA and deduced amino acid sequence of a cMnSOD from Litopenaeus vannamei were determined. The coding sequence predicts a 287 residues protein with a unique 61 amino acids extension at the N-terminus and lacking a mitochondrial-targeting sequence. Phylogenetic analysis clusters cMnSODs and mitochondrial MnSODs in two separate groups. cMnSOD transcripts were detected in hemocytes, heart, hepatopancreas, intestine, nervous system, muscle, pleopods and gills. Since hemocytes are key defense cells and their reactions produce superoxide radicals, the infection by white spot syndrome virus on the cMnSOD transcript levels were investigated and found to increase transiently 1h post-infection and then decrease as the viral infection progressed to levels significantly lower than uninfected controls by 12h post-infection.

Clinical significance of SEN virus infection in patients on maintenance haemodialysis.

BACKGROUND: The SEN virus (SENV) has been identified as a putative new hepatitis virus. This study was conducted to clarify the clinical significance of SENV infection in patients on haemodialysis. METHODS: A total of 189 patients on maintenance haemodialysis and 60 healthy controls were enrolled. Of the 189 patients, 154 were followed up for 2 years. SENV DNA (genotypes D and H) was measured by means of polymerase chain reaction. RESULTS: SENV infection was significantly (P=0.012) more prevalent in patients on haemodialysis (38%) than in controls (22%). SENV infection was not associated with the amount of transfusion or duration of haemodialysis, while hepatitis C virus (HCV) infection was significantly associated with both of these factors. Elevation of alanine aminotransferase was significantly associated with HCV, but not with SENV viraemia. Of the 154 patients who were followed up, 63 (41%) remained negative, 34 (22%) gained positivity, 28 (18%) lost it and 29 (19%) remained positive for SENV infection. Episodes of alanine aminotransferase elevation were recorded for 3% of the patients who incurred SENV infection and this rate was similar to that observed in patients who were continuously negative for SENV infection (5%). CONCLUSIONS: SENV infection was common in patients on haemodialysis. No evidence was obtained that suggested involvement of the hepatitis virus in the pathogenicity of SENV.

TT virus infection in acute non-A to E hepatitis in northern Thailand.

TT virus is a novel DNA virus widely distributed in the general population. We examined the prevalence of TTV infection in a population with acute non-A to E hepatitis and in comparison groups located in Northern Thailand. The prevalence of TTV in subjects with non-A-E hepatitis was 19% (21/112), 6% (4/72) in healthy volunteers, 17% (12/72) in those with hepatitis A or B, and 17% (8/48) in hospitalized patients with non-hepatitis illnesses. A significant association with TTV infection and non-A-E hepatitis was seen in all groups (OR 3.9, p = 0.02) and in children (OR 25.8, p = 0.001). Among subjects with non-A-E hepatitis, TTV was associated with higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (significant for AST, p = 0.02). Our observations suggest that TTV in our study population may be associated with non-A-E hepatitis and that children in particular may be at risk of hepatocellular injury as a result of TTV infection.

[The significance of TT virus detection in persons with elevated alanine aminotransferase during routine medical check up and patients with liver diseases].

OBJECTIVE: To investigate the significance of the new DNA virus (TTV) infection in persons with elevated alanine aminotransferase (ALT) during routine medical check up and patients with liver diseases, and analyze its pathogenesis. METHODS: PCR was used in the detection of TTV DNA in 19 serum samples from ALT elevated persons and 41 from ALT normal persons during their routine medical check up, and 182 serum samples from patients with various kinds of liver diseases. PCR products were confirmed by RFLP. Other hepatitis viruses were simultaneously detected using routine methods. RESULTS: TTV DNA was detected in 10.53% (2/19) and 2.44% (1/41) in groups of ALT elevated and control respectively with no significant difference between their detective rates (P > 0.05). There were 9 cases with current HBV infection, 1 case with hepatitis C and 1 case with hepatitis A in group of elevated ALT. Two TTV positive cases were both superinfected with HBV. The detecting rate of TTV DNA in patients with liver diseases was 4.4%. Only one case out of 18 non A to G hepatitis showed TTV DNA in serum. CONCLUSION: There were TTV infections in Chinese population. The main cause for ALT elevation in persons during routine medical check up was HBV infection. There were TTV infections in patients with liver diseases too. However, TTV wasn't the main cause of non A to G hepatitis. Thus, the pathogenesis of TTV might be very weak.