Jiangfeng Lan

Transcriptomic analysis of Mandarin fish brain cells infected with infectious spleen and kidney necrosis virus with an emphasis on retinoic acid-inducible gene 1-like receptors and apoptosis pathways.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant economic losses in the cultured Mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie the pathogenesis of the viral infection remain poorly understood. In this study, deep RNA sequencing technique was used to analyze the transcriptomic profiles of Mandarin fish brain cells (CPB) at progressive time points after ISKNV infection. A total of 96,206,040 clean data from 98,235,240 sequence reads were obtained. These raw data were assembled into 66,787 unigenes. Among these unigenes, 33,225 and 29,210 had significant hit the Nr and SwissProt databases where they matched 27,537and 19,638 unique protein accessions, respectively. In the samples harvested at 24 or 72 h post of the infection, a total of 10,834 or 7584 genes were differentially expressed in infected CPB cells compared to non-infected cells, including 5445 or 3766 up-regulated genes and 5389 or 3818 down-regulated genes, respectively. In addition, 12 differentially expressed genes (DEGs) were validated by quantitative PCR. These DEGs were involved in many pathways of viral pathogenesis. Further analysis of the major DEGs genes involved in the RLRs and apoptosis pathways revealed some interesting findings. In the RLRs pathway, ISKNV infection inhibited the activation of NF-κB via over expression of the IKKB-α and IKKB-β and lessened expression of interleukin-1 receptor-associated kinase 4 (IRAK4). In the apoptosis pathway, ISKNV infection could induce apoptosis mainly via tumor necrosis factor (TNF) mediated extrinsic pathway. The cellular apoptosis induced by ISKNV infection was confirmed using annexinV-FITC/PI and DAPI staining methods.

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

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.

Transcriptomic analysis of liver from grass carp (Ctenopharyngodon idellus) exposed to high environmental ammonia reveals the activation of antioxidant and apoptosis pathways

Abstract High concentration of ammonia in aquatic system leads to detrimental effects on the health of aquatic animals. However, the mechanism underlying ammonia‐induced toxicity is still not clear. To better understand the mechanism of ammonia toxicity effects on fish, juvenile grass carp was employed in the present study. RNA high‐throughput sequencing technique was applied to analyze the total RNAs extracted from the liver of fish after 8 h post exposure to the water containing 2 mM NH4HCO3 which experimentally mimicked the high environmental ammonia (HEA). A total of 49,971,114 and 53,826,986 clean reads were obtained in control and 2 mM HEA group, respectively, in which there were 911 differently expressed genes (DEGs) including 563 up‐regulated and 348 down‐regulated genes. In addition, 10 DEGs were validated by quantitative PCR. These DEGs were involved in several pathways related with oxidative stress or apoptosis. Further analysis on oxidative stress, histopathology and cellular apoptosis in grass carp liver after HEA exposure revealed interesting findings. Increased reactive oxygen species (ROS) content and superoxide dismutase (SOD) activity together with the decreased catalase (CAT) activity were detected, which may be effected by DEGs and related pathways such as FOXO signaling pathway. The histopathology and TUNEL assays results confirmed that apoptosis was induced in liver when fish had suffered HEA. Combined with the results of transcriptomic experiments, c‐Myc‐Bax‐Caspase9 apoptosis pathway could be involved in grass carp liver apoptosis induced by ammonia stress. HighlightsRNA‐sequencing was first applied to study the hepatotoxicity induced by ammonia.DEGs and phenomena about oxidative stress and apoptosis were observed in the fish.Pathways related to oxidative stress including FOXO pathway were identified.c‐Myc‐Bax‐Caspase9 apoptosis pathway was activated in fish liver by HEA exposure.

Susceptibility of Chinese Perch Brain (CPB) Cell and Mandarin Fish to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection

Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future.

A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish (Procambarus clakii)

Abstract L-type lectins are involved in glycoproteins secretory pathways and are associated with many immune responses. There is growing evidence that L-type lectins are also involved in viral replication. In this study, a novel L-type lectin (named as PcL-lectin) was identified from red swamp crayfish (Procambarus clakii). Gene sequencing and phylogenetic tree analysis results showed that the PcL-lectin was a kind of endoplasmic reticulum Golgi intermediate compartment-53 (ERGIC-53). The expression level of PcL-lectin was significantly down regulated in crayfish after challenged with white spot syndrome virus (WSSV). Recombinant PcL-lectin protein facilitated the replication of WSSV in crayfish. In addition, WSSV replication was decreased when endogenous PcL-lectin was knocked down by RNA interference in crayfish. Furthermore, PcL-lectin may interact with VP24, an envelope protein of WSSV. Our results suggest that PcL-lectin may be required for the multiplication of WSSV, and will pave a new way for the developing of strategies against WSSV infection.

GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Abstract Streptococcosis due to the bacterium Streptococcus agalactiae (S. agalactiae) has resulted in enormous economic losses in aquaculture worldwide, especially in the tilapia culture industry. Previously, there were limited vaccines that could be employed against streptococcosis in tilapia. This study aimed to develop a vaccine candidate using the glyceraldehyde‐phosphate dehydrogenase protein (GapA) of S. agalactiae encoded by the gapA gene. Tilapia were intraperitoneally injected with PBS, PBS + Freunds adjuvant, PBS + Montanides adjuvant, GapA + Freunds adjuvant, GapA + Montanides adjuvant, killed S. agalactiae whole cells (WC)+Freunds adjuvant, or killed S. agalactiae whole cells (WC)+ Montanides adjuvant. They were then challenged with S. agalactiae, and the relative percentage survival (RPS) was monitored 14 days after the challenge. The highest RPSs were observed in the WC groups, with 76.7% in WC + Freunds adjuvant and 74.4% in WC + Montanides adjuvant groups; these were followed by the GapA groups, with 63.3% in GapA + Freunds adjuvant and 45.6% in GapA + Montanides adjuvant groups. The RPS of the PBS group was 0%, and those of PBS + Freunds adjuvant and PBS + Montanides adjuvant groups were 6.7% and 3.3%, respectively. Additionally, the IgM antibody responses elicited in GapA groups and WC groups were significantly higher than those in PBS groups. Furthermore, the expressions of cytokine (IL‐1&bgr; and TNF‐&agr;) mRNAs in the GapA groups and WC groups were significantly higher than those in the PBS groups. Taken together, these results reveal that the GapA protein is a promising vaccine candidate that could be used to prevent streptococcosis in tilapia. HighlightsGapA of Streptococcus agalactiae was expressed and purified in this study.The GapA subunit vaccine can provide protection against Streptococcus agalactiae infection in tilapia.Freund’s adjuvant might be better for tilapia vaccines compared with Montanide’s adjuvant.

Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics.

Autophagy induced by snakehead fish vesiculovirus inhibited its replication in SSN-1 cell line

Autophagy plays an important role in host protection against pathogen infection through activating innate and adaptive immunity. In the present study, we observed that the infection of snakehead fish vesiculovirus (SHVV) could induce apparent autophagy in striped snakehead fish cell line (SSN-1), including clear double-membrane vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3B (SSN-LC3B) and the conversion of SSN-LC3B-Ⅰ to SSN-LC3B-Ⅱ. Furthermore, we verified that autophagy inhibited the replication of SHVV by assessing mRNA and protein level of nucleoprotein as well as virus titer in the supernatant. These results will shed a new light on the prevention of the infection of SHVV.

Co-option of bacteriophage lysozyme genes by bivalve genomes

Eukaryotes have occasionally acquired genetic material through horizontal gene transfer (HGT). However, little is known about the evolutionary and functional significance of such acquisitions. Lysozymes are ubiquitous enzymes that degrade bacterial cell walls. Here, we provide evidence that two subclasses of bivalves (Heterodonta and Palaeoheterodonta) acquired a lysozyme gene via HGT, building on earlier findings. Phylogenetic analyses place the bivalve lysozyme genes within the clade of bacteriophage lysozyme genes, indicating that the bivalves acquired the phage-type lysozyme genes from bacteriophages, either directly or through intermediate hosts. These bivalve lysozyme genes underwent dramatic structural changes after their co-option, including intron gain and fusion with other genes. Moreover, evidence suggests that recurrent gene duplication occurred in the bivalve lysozyme genes. Finally, we show the co-opted lysozymes exhibit a capacity for antibacterial action, potentially augmenting the immune function of related bivalves. This represents an intriguing evolutionary strategy in the eukaryote–microbe arms race, in which the genetic materials of bacteriophages are co-opted by eukaryotes, and then used by eukaryotes to combat bacteria, using a shared weapon against a common enemy.