Yongcan Zhou

Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including β-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.

Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus

ABSTRACT The probiotic properties of Bacillus subtilis HAINUP40 isolated from the aquatic environment, and the effects of dietary administration of B. subtilis HAINUP40 on the growth performance, intestinal probiotic recovery, digestive enzyme activities, innate immunity and disease resistance of tilapia (Oreochromis niloticus) were evaluated. The probiotic properties investigated include tolerance to simulated gastrointestinal stress, auto‐aggregation, cell surface hydrophobicity and extracellular enzyme production. The cell number of B. subtilis changed little after 4 h in simulated gastric fluid at pH = 2.0, 3.0, 4.0 and simulated intestinal fluid at pH = 6.8.B.subtilis HAINUP40 revealed strong auto‐aggregation property (34.6–87.0%) after 24 h incubation period. It exhibited significant cell surface hydrophobicity in xylene (28.8%) and chloroform (41.3%) and produced extracellular proteases and amylase. After tilapia (mean weight = 95 ± 8 g) were fed with a diet containing 108 cfu/g B. subtilis HAINUP40, their final body weight, percent weight gain (PWG), specific growth rate (SGR), total antioxidant capacity (T‐AOC) and serum superoxide dismutase (SOD) increased significantly (p < 0.05) after 8 weeks; feed conversion rate (FCR) is significantly lower (p < 0.05) after 8 weeks; the protease and amylase activity in the digestive tract increased significantly (p < 0.05) after 4 and 8 weeks; and respiratory bursts and serum lysozyme activity increased significantly (p < 0.05) after 2 weeks. Moreover, being challenged with pathogenic Streptococcus agalactiae for 2 weeks, the relative percent survival (RPS%) is 52.94%. The results of this study strongly suggest that dietary supplement of B. subtilis HAINUP40 can effectively enhances the growth performance, immune response, and disease resistance of Nile tilapia. HIGHLIGHTSBacillus subtilis HAINUP40 can tolerate conditions mimicking the gastrointestinal tract.Bacillus subtilis HAINUP40 can enhance the growth performance of tilapia.Bacillus subtilis HAINUP40 can improve the immune responses of tilapia.

Molecular cloning and characterization of two types of IκBα orthologues in orange-spotted grouper, Epinephelus coioides

Inhibitors of kappa B (IκBs) are the members of primary regulators of NF-κB, which can inhibit NF-κB activity by blocking the NF-κB in an inactive state in the cytoplasm. In this study, two types of IκBα orthologues (EcIκBαA and EcIκBαB) from orange-spotted grouper, Epinephelus coioides, were cloned and characterized. EcIκBαA and EcIκBαB encoded putative proteins containing 308 and 318 amino acids, which shared 59% and 53% identity to IκBαA and IκBαB of Danio rerio, respectively. Amino acid sequence alignment showed that both EcIκBαA and EcIκBαB contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcIκBαA and EcIκBαB contained 5 and 6 ankyrin repeats, respectively. The genomic DNA of EcIκBαA and EcIκBαB consisted of 6 exons and 5 introns. Both of their transcripts were widely distributed in different tissues, and the expression levels were different in response to various stimuli, including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Dual-luciferase reporter assay suggested that both EcIκBαA and EcIκBαB were able to inhibit Ecc-Rel and Ecp65 induced NF-κB promoter activity in grouper spleen (GS) cells. Subcellular localization analysis showed that EcIκBαB was present predominantly in the cytoplasm, while EcIκBαA was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcIκBαA and EcIκBαB in GS cells inhibited the viral gene transcriptions of MCP, ORF019 and ORF162 of SGIV. Taken together, our findings suggested that both EcIκBαA and EcIκBαB were involved in grouper innate immunity against virus.

Selection and identification of Singapore grouper iridovirus vaccine candidate antigens using bioinformatics and DNA vaccination

In this study, we described a rapid and efficient method which integrated the bioinformatic prediction and DNA vaccine technology to identify vaccine candidates against Singapore grouper iridovirus (SGIV). The 162 previously defined open reading frames (ORFs) of SGIV were subjected to extensive sequence similarity searches, as well as motif, cellular location, and domain prediction. Based on our analysis, 13 genes were chosen and cloned into the eukaryotic expression vector pcDNA 3.1. In vitro and in vivo expression of these DNA vaccine constructs was examined in Epinephelus akaara spleen cells (EAGS) and immunized fish by Western blot and RT-PCR analysis, respectively. Three weeks after the second booster, immunized fish were challenged with SGIV and the level of protection and survival was assessed. Fish vaccinated with plasmid DNA encoding viral ORF072, ORF039 and ORF036 (designated as pcDNA-72, pcDNA-39 and pcDNA-36, respectively) exhibited 66.7%, 66.7% and 58.3% relative percent survival rates, respectively, in comparison with the control fish. These three DNA vaccines induced innate immune responses, raising significantly high level of Mx expression relative to the fish vaccinated with the empty plasmid at 3 days post-vaccination. Furthermore, recombinant protein from ORF072 was also used to immunize another set of fish and similar protective effect was obtained. Taken together, our results validated the applicability of bioinformatics in genome mining, resulting in the identification of three protective antigens. The promising results obtained in the present study have prompted further testing to improve the immunogenicity of these potential DNA vaccines.

Transcriptomic profiles of striped snakehead fish cells (SSN-1) infected with red-spotted grouper nervous necrosis virus (RGNNV) with an emphasis on apoptosis pathway

ABSTRACT Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. However, the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood. In this study, the transcriptomic profiles of striped snakehead fish (Channa striatus) cells (SSN‐1) infected with red‐spotted grouper NNV (RGNNV) were investigated using deep RNA sequencing technique. From 254,955,234 raw reads, a total of 253,338,544 clean reads were obtained and they were assembled into 93,372 unigenes. Differentially expressed genes (DEGs) were identified from RGNNV‐infected or mock‐infected SSN‐1 cells, including 1184 up‐regulated and 1456 down‐regulated genes at 3 h (h) post of infection (poi), and 1138 up‐regulated and 2073 down‐regulated genes at 24 h poi, respectively. These DEGs were involved in many pathways related to viral pathogenesis, including retinoic acid‐inducible gene I (RIG‐I) like receptors pathway, apoptosis pathway, oxidative phosphorylation, PI3K‐Akt signaling pathway, and MAPK signaling pathway. Subsequent analysis focusing on the apoptosis pathway showed that the expression of Endonuclease G (EndoG) was up‐regulated upon RGNNV infection at both 3 and 24 h poi. Therefore, EndoG gene was cloned and its function was further characterized. The results showed that over‐expression of EndoG could also induce cellular apoptosis in SSN‐1 cells, indicating that RGNNV infection might induce apoptosis of SSN‐1 cells via EndoG‐associated mitochondrial pathway. These results will shed a new light on the pathogenesis of NNV. HIGHLIGHTSTranscriptomic analysis of stripped snakehead fish cell SSN‐1 infected with RGNNV.Immune‐related pathway: apoptosis pathway.EndoG is involved in the apoptosis induced by RGNNV infection in SSN‐1 cells.

Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides

A C-type lectin-like protein (Ec-CTLP) was cloned from the grouper Epinephelus coioides. The full-length cDNA of Ec-CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174-residue protein. The putative amino acid sequence of Ec-CTLP contains a signal peptide of 19 residues at the N-terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp-Asn-Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec-CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec-CTLP was distributed only in the cytoplasm. Recombinant Ec-CTLP (rEc-CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti-Ec-CTLP serum preparation. The rEc-CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram-negative E. coli and gram-positive Staphylococcus aureus) was positively correlated with the concentration of the rEc-CTLP. These findings can provide clues to help understand the probable C-type lectin in marine fish innate immunity.

JNK1 Derived from Orange-Spotted Grouper, Epinephelus coioides, Involving in the Evasion and Infection of Singapore Grouper Iridovirus (SGIV)

c-Jun N-terminal kinase (JNK) regulates cellular responses to various extracellular stimuli, environmental stresses, pathogen infections, and apoptotic agents. Here, a JNK1, Ec-JNK1, was identified from orange-spotted grouper, Epinephelus coioides. Ec-JNK1 has been found involving in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis in vitro. SGIV infection activated Ec-JNK1, of which phosphorylation of motif TPY is crucial for its activity. Over-expressing Ec-JNK1 phosphorylated transcription factors c-Jun and promoted the infection and replication of SGIV, while partial inhibition of the phosphorylation of Ec-JNK1 showed the opposite effects by over-expressing the dominant-negative EcJNK1-Δ183-185 mutant. Interestingly, SGIV enhanced the viral infectivity by activating Ec-JNK1 which in turn drastically inhibited the antiviral responses of type 1 IFN, indicating that Ec-JNK1 could be involved in blocking IFN signaling during SGIV infection. In addition, Ec-JNK1 enhanced the activation of AP-1, p53, and NF-κB, and resulted in increasing the levels of SGIV-induced cell death. The caspase 3-dependent activation correlated with the phosphorylation of Ec-JNK1 and contributed to SGIV-induced apoptosis. Taken together, SGIV modulated the phosphorylation of Ec-JNK1 to inactivate the antiviral signaling, enhance the SGIV-induced apoptosis and activate transcription factors for efficient infection and replication. The “positive cooperativity” molecular mechanism mediated by Ec-JNK1 contributes to the successful evasion and infection of iridovirus pathogenesis.

c-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis.

C-Jun N-terminal kinases (JNKs), a subgroup of serine-threonine protein kinases that activated by phosphorylation, are involve in physiological and pathophysiological processes. JNK3 is one of JNK proteins involved in JNK3 signaling transduction. In the present study, two JNK3 isoforms, Ec-JNK3 X1 and Ec-JNK3 X2, were cloned from orange-spotted grouper, Epinephelus coioides. Both Ec-JNK3 X1 and Ec-JNK3 X2 were mainly expressed in liver, gill, skin, brain and muscle of juvenile grouper. The relative expression of Ec-JNK3 X2 mRNA was much higher in muscle and gill than that of Ec-JNK3 X1. Isoform-specific immune response to challenges was revealed by the expression profiles in vivo. Immunofluorescence staining indicated that JNK3 was localized in the cytoplasm of grouper spleen (GS) cells and shown immune response to SGIV infection in vitro. Over-expressing Ec-JNK3 X1 and/or Ec-JNK3 X2 inhibited the SGIV infection and replication and the SGIV-induced apoptosis. To achieve the antiviral and anti-apoptosis activities, JNK3 promoted the activation of genes ISRE and type I IFN in the antiviral IFN signaling pathway, and inhibited the activation of transcription factors NF-κB and p53 relating to apoptosis, respectively. Ec-JNK3 X2 showed stronger activities in antivirus and anti-apoptosis than that of Ec-JNK3 X1. Our results not only define the characterization of JNK3 but also reveal new immune functions and the molecular mechanisms of JNK3 on iridoviruses infection and the virus-induced apoptosis.

MHC class IIα polymorphism and its association with resistance/susceptibility to Vibrio harveyi in golden pompano (Trachinotus ovatus)

&NA; The major histocompatibility complex (MHC) plays an important role in the vertebrate immune response to antigenic peptides, and it is essential for recognizing foreign pathogens in organisms. In this study, MHC class II&agr; (Trov‐MHC II&agr;) from the golden pompano (Trachinotus ovatus) was first cloned and identified. The gene structure of Trov‐MHC II&agr; was contained four exons and three introns. High levels of polymorphism were found in the exon 2 of Trov‐MHC II&agr;. A total of 29 different MHC class II&agr; alleles with high polymorphism were identified from 80 individuals. The ratio of non‐synonymous substitutions (dN) to synonymous substitutions (dS) was 3.157 (>1) in the peptide binding regions (PBRs) of Trov‐MHC II&agr;, suggesting positive balancing selection. Six alleles were selected to analyze the association between alleles and resistance/susceptibility to Vibrio harveyi in golden pompano. The results showed that Trov‐DAA*6401 and Trov‐DAA*6702 alleles were associated with the resistance to V. harveyi in golden pompano, while alleles Trov‐DAA*6304 and Trov‐DAA*7301 were associated with the susceptibility to V. harveyi in golden pompano. This study confirmed the association between alleles of MHC class II&agr; and disease resistance, and also detected some alleles which might be correlated with high V. harveyi‐resistance. These disease resistance‐related MHC alleles could be used as potential genetic markers for molecular marker‐assisted selective breeding in the golden pompano. HighlightsWe first cloned and identified MHC class II&agr; (Trov‐MHC II&agr;) from the golden pompano (Trachinotus ovatus).We found high polymorphism in the exon 2 of Trov‐MHC II&agr;.Trov‐DAA*6401 and Trov‐DAA*6702 alleles were showed associated with the resistance to V. harveyi in golden pompano.Trov‐DAA*6304 and Trov‐DAA*7301 alleles were showed associated with the susceptibility to V. harveyi in golden pompano.

Differential expression of HPG-axis genes in autotetraploids derived from red crucian carp Carassius auratus ♀ × blunt snout bream Megalobrama amblycephala, ♂

Autotetraploid fish (4n = 200, abbreviated as 4nRR), which reach sexual maturity at 1 year of age, were derived from the whole genome duplication of red crucian carp Carassius auratus red var. (RCC; 2n = 100) and possess four sets of chromosomes from RCC. The histological features of the gonads showed that the RCC and 4nRR both possessed normal gonadal structure and could arrive at maturation. To understand the expression characteristics of genes related to reproductive development in the autotetraploid fish, we analysed the nucleotide sequence and expression characteristics of the gnrh2, gthb and gthr genes, which are the pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis. We found that the gnrh2, gthb and gthr genes in 4nRR share remarkable homology with RCC, but there were obvious differences in expression levels between 4nRR and RCC. These results demonstrate that autotetraploidization can lead to gene expression changes. This study provides insights into the molecular mechanism underlying the reproductive development of autotetraploid fish and is expected to be of great significance for subsequent research on polyploidization.