The zebrafish as a potential model for vaccine and adjuvant development.

INTRODUCTION: Zebrafishes represent a proven model for human diseases and systems biology, exhibiting physiological and genetic similarities and having innate and adaptive immune systems. However, they are underexplored for human vaccinology, vaccine development, and testing. Here we summarize gaps and challenges. AREAS COVERED: Zebrafish models have four potential applications: 1) Vaccine safety: The past successes in using zebrafishes to test xenobiotics could extend to vaccine and adjuvant formulations for general safety or target organs due to the zebrafish embryos' optical transparency. 2) Innate immunity: The zebrafish offers refined ways to examine vaccine effects through signaling via Toll-like or NOD-like receptors in zebrafish myeloid cells. 3) Adaptive immunity: Zebrafishes produce IgM, IgD,and two IgZ immunoglobulins, but these are understudied, due to a lack of immunological reagents for challenge studies. 4) Systems vaccinology: Due to the availability of a well-referenced zebrafish genome, transcriptome, proteome, and epigenome, this model offers potential here. EXPERT OPINION: It remains unproven whether zebrafishes can be employed for testing and developing human vaccines. We are still at the hypothesis-generating stage, although it is possible to begin outlining experiments for this purpose. Through transgenic manipulation, zebrafish models could offer new paths for shaping animal models and systems vaccinology.

LGP2 Facilitates Bacterial Escape through Binding Peptidoglycan via EEK Motif and Suppressing NOD2-RIP2 Axis in Cyprinidae and Xenocyprididae Families.

RIG-I-like receptors and NOD-like receptors play pivotal roles in recognizing microbe-associated molecular patterns and initiating immune responses. The LGP2 and NOD2 proteins are important members of the RIG-I-like receptor and NOD-like receptor families, recognizing viral RNA and bacterial peptidoglycan (PGN), respectively. However, in some instances bacterial infections can induce LPG2 expression via a mechanism that remains largely unknown. In the current study, we found that LGP2 can compete with NOD2 for PGN binding and inhibit antibacterial immunity by suppressing the NOD2-RIP2 axis. Recombinant CiLGP2 (Ctenopharyngodon idella LGP2) produced using either prokaryotic or eukaryotic expression platform can bind PGN and bacteria in pull-down and ELISA assays. Comparative protein structure models and intermolecular interaction prediction calculations as well as pull-down and colocalization experiments indicated that CiLGP2 binds PGN via its EEK motif with species and structural specificity. EEK deletion abolished PGN binding of CiLGP2, but insertion of the CiLGP2 EEK motif into zebrafish and mouse LGP2 did not confer PGN binding activity. CiLGP2 also facilitates bacterial replication by interacting with CiNOD2 to suppress expression of NOD2-RIP2 pathway genes. Sequence analysis and experimental verification demonstrated that LGP2 having EEK motif that can negatively regulate antibacterial immune function is present in Cyprinidae and Xenocyprididae families. These results show that LGP2 containing EEK motif competes with NOD2 for PGN binding and suppresses antibacterial immunity by inhibiting the NOD2-RIP2 axis, indicating that LGP2 plays a crucial negative role in antibacterial response beyond its classical regulatory function in antiviral immunity.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

Effects of Lactobacillus acidophilus AC on the growth, intestinal flora and metabolism of zebrafish (Danio rerio).

The intensive aquaculture model has resulted in a heightened prevalence of diseases among farmed animals. It is imperative to identify healthy and efficacious alternatives to antibiotics for the sustainable progression of aquaculture. In this investigation, a strain of Lactobacillus acidophilus AC was introduced into the cultural water at varying concentrations (105 CFU/mL, 106 CFU/mL, 107 CFU/mL) to nourish zebrafish (Danio rerio). The findings revealed that L. acidophilus AC effectively increased the growth performance of zebrafish, improved the ion exchange capacity of gills, and enhanced hepatic antioxidant and immune-enzyme activities. Furthermore, L. acidophilus AC notably enhanced the intestinal morphology and augmented the activity of digestive enzymes within the intestinal tract. Analysis of intestinal flora revealed that L. acidophilus AC exerted a significant impact on the intestinal flora community, manifested by a reduction in the relative abundance of Burkholderiales, Candidatus_Saccharibacteria_bacterium, and Sutterellaceae, coupled with an increase in the relative abundance of Cetobacterium. Metabolomics analysis demonstrated that L. acidophilus AC significantly affected intestinal metabolism of zebrafish. PG (i-19:0/PGE2) and 12-Hydroxy-13-O-d-glucuronoside-octadec-9Z-enoate were the metabolites with the most significant up- and down-regulation folds, respectively. Finally, L. acidophilus AC increased the resistance of zebrafish to Aeromonas hydrophila. In conclusion, L. acidophilus AC was effective in enhancing the health and immunity of zebrafish. Thus, our findings suggested that L. acidophilus AC had potential applications and offered a reference for its use in aquaculture.

Okadaic acid enhances NfKB, TLR-4, caspase 3, ERK ½, c-FOS, and 8-OHdG signaling pathways activation in brain tissues of zebrafish larvae.

This study was designed to investigate the potential neuronal damage mechanism of the okadaic acid (OA) in the brain tissues of zebrafish embryos by evaluating in terms of immunofluorescence of Nf KB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG signaling pathways. We also evaluated body malformations. For this purpose, zebrafish embryos were exposed to 0.5 µg/ml, 1 µg/ml and 2.5 µg/ml of OA for 5 days. After application, FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG respectively. The results indicated that OA caused immunofluorescence positivity of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG in a dose-dependent manner in the brain tissues of zebrafish embryos. Pericardial edema (PE), nutrient sac edema (YSE) and body malformations, tail malformation, short tail and head malformation (BM) were detected in zebrafish embryos. These results suggest that OA induces neuronal damage by affecting the modulation of DNA damage, apoptotic, and inflammatory activities in the brain tissues of zebrafish embryos. The increase in signaling pathways shows that OA can cause damage in the structure and function of brain nerve cells. Our results provide a new basis for the comprehensive assessment of the neural damage of OA and will offer enable us to better understand molecular the mechanisms underlying the pathophysiology of OA toxicity.

Fish ubiquitin-specific protease 8 (USP8) inhibits IFN production through autophagy-lysosomal dependent degradation of IRF7.

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response.

Carbamazepine transmits immune effect by activation of gut-liver axis and TLR signaling pathway from parental zebrafish to offspring.

Carbamazepine (CBZ) has been identified in the aquatic environment as an emerging contaminant. Its immune effect across generations at environmentally relevant concentrations is little known. We aim to elucidate the effects of CBZ on the immune system in zebrafish (Danio rerio), hypothesizing the effects caused by CBZ exposure in the parental generation can be passed on to its offspring, leading to impairment of innate immune function and defense against pathogen weakened. A suite of bioassays (including a test with added lipopolysaccharide) was used to measure the effects of environmentally relevant levels of CBZ (1, 10, and 100 µg/l) on zebrafish at multiple biological levels, and across 2 successive generations (21 days exposure for F0; 5 and 21 days exposure or nonexposure for F1). The results showed that CBZ affected homeostasis in the immune system, caused liver vacuolization, increased the inflammation-related microbiota proportion in gut, and decreased reproduction, by induction of oxidative stress and modulation of Toll-like receptors (TLR) signaling pathway on gut-liver axis. The effects of exposure to CBZ over 21 days in F0 could be passed to the next generation. Intergenerational effects on TLR and antioxidant defense system were also observed in nonexposed F1 at 5 days post-fertilization (5 dpf), but diminished at 21 dpf. The finding provided evidence to unravel immune response by gut-liver axis mediated and oxidative stress under 4 test conditions. The study has raised a potential concern about the multigenerational immune effects of environmental pollutants and calls for a focus on the risk of synergetic pathogen infection.

A zebrafish NLRX1 isoform downregulates fish IFN responses by targeting the adaptor STING.

In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.

Zebrafish TMEM47 is an effective blocker of IFN production during RNA and DNA virus infection.

IMPORTANCE: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

Cd248a and Cd248b in zebrafish participate in innate immune responses.

CD248, also known as endosialin or tumor endothelial marker 1, is a type I single transmembrane glycoprotein. CD248 has been demonstrated to be upregulated in cancers, tumors and many fibrotic diseases in human and mice, such as liver damage, pulmonary fibrosis, renal fibrosis, arthritis and tumor neovascularization. However, no definite CD248 orthologs in fish have been documented so far. In this study, we report the identification of cd248a and cd248b in the zebrafish. Both the phylogenetic analysis and the conserved synteny strongly suggested that zebrafish cd248a and cd248b are orthologs of the human CD248. Both cd248a and cd248b exhibited similar and dynamic expression pattern in early development, both genes had weak maternal expression, the zygotic transcripts were first seen in anterior somites and head mesenchyme, then shifted to eyes and head mesenchyme, later expanded to branchial arches, and gradually declined with development. The expression profiles of cd248a and cd248b were upregulated upon LPS (Lipopolysaccharide) challenge. Both Cd248a protein and Cd248b protein were localized on the cell membrane and cytoplasm, and overexpression of cd248a and cd248b induced the expression of pro-inflammatory cytokines, in vitro and in vivo. Moreover, deficiency of cd248a or cd248b both downregulated the expression of pro-inflammatory cytokines and upregulated anti-inflammatory cytokine. Additionally, loss of cd248a or cd248b both downregulated the expression of pro-inflammatory cytokines after LPS treatment. Taken together, these results indicated that cd248a and cd248b in zebrafish were involved in immune response and would provide further information to understand functions of Cd248 protein in innate immunity of fish.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Stabilized fermentation product of Cetobacterium somerae improves gut and liver health and antiviral immunity of zebrafish.

Probiotics are widely used in aquafeeds and exhibited beneficial effects on fish by improving host health and resisting pathogens. However, probiotics applied to aquaculture are mainly from terrestrial sources instead of the host animal. The purpose of the work was to evaluate the effects of stabilized fermentation product of commensal Cetobacterium somerae XMX-1 on gut, liver health and antiviral immunity of zebrafish. A total of 240 zebrafish were assigned to the control (fed a basal diet) and XMX-1 group (fed a basal diet with 10 g XMX-1/kg diet). After four weeks feeding, growth performance, feed utilization, hepatic steatosis score, TAG, lipid metabolism related genes and serum ALT were evaluated. Furthermore, serum LPS, the expression of Hif-1α, intestinal inflammation score, antioxidant capability and gut microbiota were tested. The survival rate and the expression of antiviral genes were analyzed after challenge by spring viremia of carp virus (SVCV). Results showed that dietary XMX-1 did not affect growth of zebrafish. However, dietary XMX-1 significantly decreased the level of serum LPS, intestinal inflammation score and intestinal MDA, as well as increased T-AOC and the expression of Hif-1α in zebrafish intestine (p < 0.05). Furthermore, XMX-1 supplementation decreased the relative abundance of Proteobacteria and increased Firmicutes and Actinobacteria. Additionally, XMX-1 supplementation significantly decreased hepatic steatosis score, hepatic TAG, serum ALT and increased the expression of lipolysis genes versus control (p < 0.05). Zebrafish fed XMX-1 diet exhibited higher survival rate after SVCV challenge. Consistently, dietary XMX-1 fermentation product increased the expression of IFNφ2 and IFNφ3 after 2 days of SVCV challenge and the expression of IFNφ1, IFNφ2 and MxC after 4 days of SVCV challenge in the spleen in zebrafish versus control (p < 0.05). In conclusion, our results indicate that dietary XMX-1 can improve liver and gut health, while enhancing antiviral immunity of zebrafish.

Teleost Fish as an Experimental Model for Vaccine Development.

Advances in vaccine development depend on animal models to test innovative therapies. Recent studies have reported the successful introduction of teleost fish as a new vertebrate model in scientific research, with emphasis on the species Danio rerio (zebrafish). This chapter aims to give an overview of important aspects related to the immune system of fish, as well as the current progress of the successful use of these animals in studies for the development of vaccines, assisting in the determination of efficacy and clinical safety. Among the advantages of using fish for the development of vaccines and immunomodulatory drugs, it is worth highlighting the reproductive capacity of these animals resulting in a high number of individuals belonging to the same spawning, transparent embryos, low cost of breeding and high genetic similarity that favor translational responses to vertebrate organisms like humans.

SCFAs improve disease resistance via modulate gut microbiota, enhance immune response and increase antioxidative capacity in the host.

To evaluate the effects of dietary short chain fatty acids (SCFAs) on the intestinal health and innate immunity in crucian carp, a six-week feeding trial was carried out with following treatments: basal diet (BD), basal diet supplementation with 1% sodium acetate (BDSA), basal diet supplementation with 1% sodium propionate (BDSP) and basal diet supplementation with 1% sodium butyrate (BDSB). The results showed dietary BDSA, BDSP and BDSB could protect the host against oxidative stress by improving the activity of certain antioxidative enzymes (T-SOD, GSH-Px and CAT). Additionally, dietary SCFAs could enhance mucosal and humoral immune responses by improving certain innate immune parameters in serum and skin mucus productions (IgM, ACH50 and T-SOD). Furthermore, dietary BDSA and BDSP could up-regulate the expression of immune related genes (TNF-α, TGF-ß and IL-8) and tight junction protein genes (occludin and ZO-1). Dietary BDSB could also elevate the expression of IL-8, TGF-ß, ZO-1 and Occludin in the midgut. Although dietary differences of SCFAs didn't alter the α-diversity of the intestinal flora, they altered the core microbiota. Finally, the challenge trial showed that dietary basal diet supplementation with SCFAs could protect zebrafish against Aeromonas hydrophila. These results suggest that dietary SCFAs could improve innate immunity, modulate gut microbiota and increase disease resistance in the host, which indicated the potential of SCFAs as immunostimulants in aquaculture.

Cross-species opsonic activity of zebrafish fish-egg lectin on mouse macrophages.

Zebrafish Fish-egg lectin (zFEL) has been identified and proved to be a maternal factor with antibacterial and opsonic ability in fishes. In this study, we found that zFEL was capable of enhancing the phagocytosis of the bacteria by macrophages of mouse (RAW264.7 and mouse peritoneal macrophages), suggesting a cross-species function of zFEL in higher animals. Further studies showed that zFEL can active the antigen presentation ability by up-regulating the expression of CD80, CD86 and MHC II. Meanwhile, zFEL also promoted the polarization of macrophages to M1-type, which was confirmed by the increase of cytokines TNF-α and IL-6. The expression of p38 gene was up-regulated in macrophages preincubated with zFEL. Taken together, zFEL appears opsonic function in mammal macrophages and has potential application in immunomodulation.

Retinal ganglion cell survival after severe optic nerve injury is modulated by crosstalk between Jak/Stat signaling and innate immune responses in the zebrafish retina.

Visual information is transmitted from the eye to the brain along the optic nerve, a structure composed of retinal ganglion cell (RGC) axons. The optic nerve is highly vulnerable to damage in neurodegenerative diseases, such as glaucoma, and there are currently no FDA-approved drugs or therapies to protect RGCs from death. Zebrafish possess remarkable neuroprotective and regenerative abilities. Here, utilizing an optic nerve transection (ONT) injury and an RNA-seq-based approach, we identify genes and pathways active in RGCs that may modulate their survival. Through pharmacological perturbation, we demonstrate that Jak/Stat pathway activity is required for RGC survival after ONT. Furthermore, we show that immune responses directly contribute to RGC death after ONT; macrophages/microglia are recruited to the retina and blocking neuroinflammation or depleting these cells after ONT rescues survival of RGCs. Taken together, these data support a model in which crosstalk between macrophages/microglia and RGCs, mediated by Jak/Stat pathway activity, regulates RGC survival after optic nerve injury.

A Comprehensive Annotation of the Channel Catfish (Ictalurus punctatus) T Cell Receptor Alpha/Delta, Beta, and Gamma Loci.

The complete germline repertoires of the channel catfish, Ictalurus punctatus, T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.

Activation of the TCA Cycle to Provide Immune Protection in Zebrafish Immunized by High Magnesium-Prepared Vibrio alginolyticus Vaccine.

Vaccines are safe and efficient in controlling bacterial diseases in the aquaculture industry and are in line with green farming. The present study develops a previously unreported approach to prepare a live-attenuated V. alginolyticus vaccine by culturing bacteria in a high concentration of magnesium to attenuate bacterial virulence. Furthermore, metabolomes of zebrafish immunized with the live-attenuated vaccines were compared with those of survival and dying zebrafish infected by V. alginolyticus. The enhanced TCA cycle and increased fumarate were identified as the most key metabolic pathways and the crucial biomarker of vaccine-mediated and survival fish, respectively. Exogenous fumarate promoted expression of il1ß, il8, il21, nf-κb, and lysozyme in a dose-dependent manner. Among the five innate immune genes, the elevated il1ß, il8, and lysozyme are overlapped in the vaccine-immunized zebrafish and the survival from the infection. These findings highlight a way in development of vaccines and exploration of the underlying mechanisms.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.