Cellular metabolism has been hijacked by viruses for efficient replication due to their regulatory roles on both innate and adaptive immunity. Largemouth bass virus (LMBV), a member of Ranavirus, causes high mortality and severe economic losses in several fish species, seriously threatening the sustainable development of aquaculture industry. However, the roles of the metabolic events on LMBV replication remained largely uncertain. Here, our results showed that LMBV boosted aerobic glycolysis both in vitro and in vivo. Supplement with glucose significantly enhanced LMBV replication in vitro. Consistently, blocking glycolysis with 2DG or lactate biosynthesis with oxamate markedly decreased LMBV replication, as indicated by reduced viral gene expression, protein synthesis, and virus titers. Interestingly, exogenous lactate also significantly promoted LMBV replication. Mechanistically, lactate not only reduced interferon response triggered by poly(I:C), but also promoted cholesterol biosynthesis. Further analysis showed that lactate induced cholesterol biosynthesis might inhibit LMBV induced IRF3 phosphorylation, and reduce the expression levels of IFN-stimulated genes (e.g., IFN1, IFNγ, IRF3, IRF7, ISG20, MX). Thus, we speculated that lactate might facilitate LMBV replication by enhancing cholesterol biosynthesis and inhibiting host interferon response. Our results provided new insights into understanding the roles of metabolic reprogramming during iridovirus infection.

ATP-binding cassette (ABC) transporters play crucial roles in stress responses across organisms, yet their functions in Fenneropenaeus chinensis remain largely unknown. In this study, we identified 42 FcABC genes (FcABCs) in the F. chinensis genome and analyzed their phylogenetic relationships, gene structures, and chromosomal distributions. Phylogenetic analysis grouped the FcABCs into eight subfamilies (ABCA-ABCH), with conserved motif and domain compositions within each subfamily. Expression analysis showed that several FcABC genes, including FcABCG5, FcABCA1, and FcABCC3, were significantly induced under saline-alkaline stress in gill and hepatopancreas tissues. In contrast, most FcABCs were downregulated after WSSV challenge, though a subset (e.g., FcABCB1, FcABCC1) exhibited early upregulation. Functional validation via RNA interference demonstrated that knockdown of FcABCG5 increased shrimp mortality under saline-alkaline stress. Cis-regulatory element analysis revealed an enrichment of stress- and immune-related elements in FcABC promoters. Protein-protein interaction network predictions indicated potential roles for FcABCs in cholesterol metabolism and organic anion transport. Our findings provide insights into the roles of FcABC genes in stress adaptation and immune defense, offering candidate genes for the breeding of stress-resistant shrimp varieties.

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a vital factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system. The structure and function of IRAK1BP1 have been well investigated in mammals, yet the piscine IRAK1BP1 is poorly understood. In the present study, the first teleost fish IRAK1BP1 from blunt snout bream, maIRAK1BP1, was identified and characterized, which was composed of a 750 bp open reading frame encoding a protein of 249 amino acids. Phylogenetic analysis indicated that maIRAK1BP1, is a novel member of IRAK1BP1 family and showed the highest similarity to that of grass carp (96.28%). Amino acid sequence analysis revealed that maIRAK1BP1 shared a conserved SIMPL domain. Spatial expression pattern analysis revealed that maIRAK1BP1 was constitutively expressed in various normal tissues including heart, spleen, head kidneys, gill, kidney, muscle, intestine and liver. The maIRAK1BP1 expression in immune-relevant tissues (liver, head kidneys and kidney) and caudal fin cells (MAF) were significantly up-regulated after LPS and bacteria injection. The result from subcellular localization analysis indicated that maIRAK1BP1 protein was localized in both the cytoplasm and nucleus of MAF cells, but predominantly existed in cytoplasm. The activity of NF-κB promoter could be inhibited by overexpression of maIRAK1BP1 in HEK-293T cells. Overexpression of maIRAK1BP1 in MAF cells inhibited dramatically the transcriptional expression level of lipopolysaccharide-induced proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). In contrast, an increased expression of these cytokines was observed when maIRAK1BP1 was knocked down via siRNA. Taken together, these results indicated that maIRAK1BP1 played an important role in mediating host innate immune response to pathogen invasion, and disclosed the involvement of maIRAK1BP1 in NF-κB-mediated production of proinflammatory cytokines, which provided foundational insights into the conserved role of IRAK1BP1 in teleost immune regulation.

The tripartite motif-containing (TRIM) protein family is crucial for antiviral innate immunity. In teleost, finTRIM proteins (FTRs) represent a fish-specific TRIM subfamily that underwent extensive lineage-specific expansion. Nevertheless, the biological roles of many FTR members have yet to be fully deciphered. Here, we identified common carp finTRIM54 (CcFTR54) as potent restriction factor against spring viremia of carp virus (SVCV) infection by targeting the viral phosphoprotein (P). CcFTR54 overexpression significantly rescued the type I interferon (IFN-I) signaling from SVCV P-mediated suppression. Mechanistic investigations revealed that CcFTR54 directly bond to P protein via its PRY/SPRY domain, enhancing the recruitment of the cargo receptor NBR1 to the P protein. Consequently, NBR1 deficiency substantially attenuated both CcFTR54-mediated P protein degradation and its antiviral efficacy. Furthermore, CcFTR54 catalyzes K6- and K33-linked ubiquitination of P protein at K205, a site crucial for SVCV replication. Together, our findings unveil a novel mechanism where CcFTR54 restricts SVCV replication via NBR1-dependent selective autophagic degradation of the viral P protein, thereby broadening our insights into finTRIM-mediated antiviral immunity in teleosts.

NK-lysin, a member of the saposin-like protein family, is a potent antimicrobial peptide that plays a key role in host defense against various pathogens. However, research on its antiviral activity and underlying mechanisms remains limited. In this study, the anti-SVCV activity and action mechanism of NKLj27, an NK-lysin derived peptide from Lateolabrax japonicus, were investigated primarily in EPC cells. Cytotoxicity assays revealed that NKLj27 was non-toxic to EPC cells at concentrations ranging from 5 μM to 160 μM. NKLj27 significantly inhibited SVCV infection in EPC cells, as demonstrated by marked reductions in viral infectivity, cytopathic effects, and plaque formation, together with decreased apoptosis and necrosis. Mechanistically, NKLj27 appears to exert its antiviral activity through a dual-target strategy, acting on both the viral particle and host cells. Further investigation revealed that NKLj27 interfered with the early stages of SVCV infection by inhibiting viral adhesion and cellular entry. Additionally, NKLj27 bound to and was partially internalized by EPC cells, and it also activated the type I interferon signaling, which was shown to play an important role in its antiviral activity through inhibition assays. Finally, in vivo experiments confirmed that administration of NKLj27 significantly reduced the mortality of SVCV-infected common carp and markedly decreased viral loads in the spleen, liver, and gill tissues. Collectively, these findings demonstrate that NKLj27 inhibits SVCV infection through a dual-target mechanism involving multiple pathways, highlighting its promising potential as a novel antiviral agent for application in aquaculture.

High-fat diets (HFDs) can impair intestinal homeostasis and innate immune function in fish, whereas nutritional immunomodulation may provide an effective strategy to improve immune response to LPS challenge in aquaculture species. This study investigated whether dietary lutein (LU) alleviates HFD-induced immune dysfunction and improves the response of juvenile black seabream (Acanthopagrus schlegelii) to lipopolysaccharide (LPS) challenge. Fish were fed a normal-fat diet containing 8% lipid, an HFD containing 18% lipid, or the HFD supplemented with 0.125%, 0.25% and 0.50% LU for 10 weeks, followed by intraperitoneal injection with phosphate-buffered saline or LPS. Compared with the HFD group, dietary LU enhanced the levels of AKP, ACP, and ALB in serum and significantly decreased the levels of ALT and AST (P < 0.05). HFD altered intestinal morphology, as reflected by increased goblet cell number and reduced villus length, whereas LU supplementation up-regulated the expression of intestinal physical barrier related genes, including oclnα, cldn1 and cldn3, and suppressed the expression levels of mucosal immune stress genes, including grp78, ire1α and xbp1 and pro-inflammatory factors (nf-κb, tnfɑ) (P < 0.05). After LPS challenge, intestinal barrier-related genes were down-regulated, whereas LPS accumulation, complement activation, tlr/myd88/nf-κb signaling, inflammatory cytokine expression and apoptosis-related genes were markedly induced in the intestine and head kidney. Notably, LU supplementation modulated pparγ/rxr-related immune signaling, inhibited excessive TLR pathway activation, and attenuated LPS-induced inflammation and apoptosis. Overall, dietary LU alleviated HFD- and LPS-induced nonspecific immune impairment in juvenile black seabream, probably by maintaining intestinal barrier integrity and regulating the pparγ/rxr-tlr/nf-κb immune signaling pathway.

Carpione rhabdovirus 2023 (CAPRV2023) is an emerging, highly pathogenic pathogen infecting Golden pompano (Trachinotus anak), capable of triggering mass mortality in marine aquaculture populations and causing severe economic losses. Berberine (BBR) and glycyrrhetinic acid (GA) are natural active compounds classified as an isoquinoline alkaloid and a pentacyclic triterpenoid, respectively, both of which are widely utilized in traditional medicine owing to their antiviral, anti-inflammatory, and hepatoprotective properties. In this study, the combined therapeutic efficacy of the BBR and GA combination against CAPRV2023 infection was systematically evaluated at both in vitro and in vivo levels, with the underlying mechanistic pathways of its protective effects further comprehensively investigated. The results demonstrated that the BBR and GA combination exhibited significant antiviral and cytoprotective activities against CAPRV2023 in a time-dependent manner. The combined administration significantly enhanced the survival rate of CAPRV2023-infected Trachinotus anak, reaching up to 63.33%, while concurrently reducing tissue viral loads markedly. This comprehensive protective effect of the combination therapy is primarily attributed to its dual regulatory mechanisms of suppressing viral replication and alleviating host tissue damage. Specifically, this is achieved by attenuating NF-κB-mediated inflammatory cascade amplification, modulating apoptosis-related stress, and regulating immune and oxidative factors including IFN1, RIG-I, Mx, IL-1β, TNF-α, Caspase-3, SOD, and MDA. Overall, these findings indicate that the BBR and GA combination may represent a promising candidate strategy for CAPRV2023 prevention and control, pending further validation, providing preliminary experimental evidence for developing candidate antiviral intervention strategies in marine aquaculture.

Nesfatin-1 is an 82-amino acid polypeptide derived from the precursor protein nucleobindin 2 (NUCB2), which is a conserved multifunctional peptide in vertebrates that plays a key role in metabolic regulation, inflammatory response and ferroptosis. Although Nesfatin-1 has been characterized in various vertebrates, from mammals to teleosts, its structural and functional characteristics in primitive vertebrates remain unclear. In this study, Nesfatin-1 orthologs from the most primitive jawless vertebrate lamprey (Lethenteron camtschaticum) were cloned and identified, designated as Lc-Nesfatin-1. Bioinformatic analysis revealed that Nesfatin-1 of lamprey is relatively conserved in both sequence similarity and three-dimensional (3D) structure compared to that of higher vertebrates. Lc-Nesfatin-1 was significantly upregulated under lipopolysaccharide (LPS) stimulation, which also markedly induced the expression of pro-inflammatory cytokines, the key signaling molecule NF-κB, and anti-inflammatory cytokines. Notably, Flow cytometry analysis showed that Lc-Nesfatin-1 did not significantly inhibit LPS-induced intracellular ROS production. Furthermore, Nesfatin-1 serves as an effective negative regulator that significantly inhibits ferroptosis. These results reveal that Lc-Nesfatin-1 plays a critical role in modulating both LPS-induced inflammatory responses and ferroptosis. This study has successfully elucidated the characteristics and functions of Nesfatin-1 in primitive vertebrates and provided valuable insights into the investigation of inflammatory responses and ferroptosis in jawless vertebrates.

RNF128, also known as GRAIL, is a well-studied E3 ubiquitin ligase involved in the induction and maintenance of T cell anergy. It contains PA_GRAIL_like domain, HRD1 domain and RING-H2_like domain. Recent studies have shown that RNF128 is involved in the regulation of innate immunity in mammals. It should be noted that zebrafish, possess two RNF128 paralogs, RNF128a and RNF128b. We focused on RNF128a, which shares the closest sequence homology with mammalian RNF128; however, its role in fish has not yet been elucidated. In this paper, we demonstrated that RNF128a acts as a positive regulator of innate immunity in Zebrafish. Zebrafish RNF128a responds to multiple stimuli including SVCV (Spring Viremia of Carp Virus), poly(I:C), B-DNA, LPS, and is characterized by the heightened sensitivity to SVCV. Notably, Zebrafish RNF128a up-regulates the expression of IFN1, ISG15, MX, as well as the inflammatory cytokines such as IL-6 and TNFα in response to SVCV. Mechanistically, Zebrafish RNF128a interacts with TBK1 through its protease-associated (PA) domain. However, the subcellular localization of RNF128a is associated with its HRD1 domain. In addition, Zebrafish RNF128a is localized to both early endosome RAB5 and late endosome RAB7 but rarely in Golgi apparatus and endoplasmic reticulum. Structurally, only full-length RNF128a promotes the K63-linked ubiquitination of TBK1, while none of the truncation mutants retains this activity. Our study identified Zebrafish RNF128a as an E3 ligase that catalyzes K63-linked ubiquitination and activates TBK1, and delineated its essential role in the antiviral innate immune response in zebrafish.

Ladderlectin, a member of the C-type lectin (CTL) family, has historically been identified only in fish and shrimp, where it is primarily involved in innate immune defense. In teleosts, ladderlectin plays a crucial dual role in both innate immunity against pathogens and reproductive regulation. However, the molecular characterization and immunomodulatory functions of ladderlectin homolog (ladderlectin-like) in Hexagrammos otakii remain poorly understood. In this study, a novel ladderlectin-like gene (HoLL-like) was identified and cloned from Hexagrammos otakii based on genomic data. Sequence analysis and domain prediction revealed that HoLL-like contains a 534-bp open reading frame encoding a 177-aa polypeptide, with an N-terminal signal peptide and a single C-type lectin-like domain (CTLD). The CTLD retains the WSD, WND, and WIGG motifs and is rich in cysteine residues. Phylogenetic analysis showed that HoLL-like forms a distinct, relatively independent clade within the teleost ladderlectin-like family. Quantitative RT-PCR analysis demonstrated that Holl-like is expressed in multiple tissues, with the highest transcript levels in the liver. Besides, it exhibited a time-dependent transcriptional response in the liver, spleen, skin, intestine, and kidney following Vibrio harveyi infection. After heterologous expression, purification, and refolding, the recombinant protein rHoLL-like displayed characteristic ladder-like oligomeric bands in immunoblotting. Functional assays revealed that rHoLL-like binds and agglutinates multiple Gram-positive and Gram-negative bacteria in a Ca[2+]-independent manner and binds pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), and poly(I:C) in a dose-dependent manner, showing higher affinity for LPS. Further analyses indicated that rHoLL-like did not directly agglutinate or lyse erythrocytes, but enhanced serum hemolytic and bactericidal activities under Ca[2+]-containing conditions. These effects were sensitive to EDTA and serum heat inactivation, suggesting that rHoLL-like may modulate heat-labile, Ca[2+]-dependent serum effector mechanisms, potentially associated with complement activity. Collectively, these findings suggest that HoLL-like plays a crucial role in the innate immune response of H. otakii against bacterial and other pathogen infections. This study expands our understanding of the functional repertoire of teleost ladderlectin-like proteins and provides new insights into the molecular strategies to prevent and control bacterial diseases in marine aquaculture, as well as for the development of immunoactive agents.

Rock bream iridovirus (RBIV) causes a rapid systemic infection, severe hematological disruption, and high mortality in rock bream (Oplegnathus fasciatus). Although β-glucan is widely used as an immunostimulant in aquaculture, its influence on RBIV disease progression remains poorly understood. Therefore, this study evaluated the effects of different β-glucan administration schedules on survival, viral dissemination, and blood-associated physiological responses during experimental RBIV infection. Among the tested schedules, the 300 μg/fish Pre + Post regimen (-2 and + 2 dpi) provided the greatest survival benefit, reducing cumulative mortality from 100% to 60%, and was selected for further analyses. Compared with virus-only fish, β-glucan treatment improved survival while delaying systemic viral dissemination, reducing viral burden, and attenuating hematological deterioration throughout infection. Attenuation analyses demonstrated stage-dependent protection, with the strongest reductions in leukocyte-associated disturbance occurring during the early phase of infection, whereas preservation of erythrocyte-associated parameters was most evident during the middle phase. In contrast, biochemical responses showed comparatively limited treatment-associated effects. Overall, these findings indicate that β-glucan did not function as a direct antiviral agent but instead modified the trajectory of RBIV disease progression by slowing systemic viral expansion and attenuating hematological deterioration. These results further support the concept that preservation of hematological integrity represents a key component of disease resilience during RBIV infection.

Members of the cystatin superfamily serve as key regulators of cysteine protease activity that play critical roles in immune defense and inflammatory regulation, yet systematic studies on this family in Japanese flounder (Paralichthys olivaceus) remain limited. In this study, five cystatin family members were identified from the P. olivaceus genome, designated as Pocystatin B, Pocystatin C, Pocystatin, Pocystatin F, and Pokininogen 1. Phylogenetic analysis, gene structure characterization, conserved motif identification, and multiple sequence alignment collectively revealed the highly conserved structural features of this family. Ka/Ks analysis demonstrated that all members were subject to purifying selection, while chromosomal collinearity analysis reflected the functional divergence pattern following whole-genome duplication in teleosts. Tissue expression analysis showed that Pocystatin B and Pocystatin C were predominantly expressed in gills and liver, respectively, suggesting their specialized physiological roles in distinct immune organs. Following Edwardsiella tarda challenge, both genes exhibited differential dynamic expression patterns in blood, kidney, and gill tissues, indicating that they cooperatively participated in innate immune responses through distinct regulatory pathways. In vitro functional assays confirmed that both rPoCystatin B and rPoCystatin C possessed broad-spectrum antibacterial activity, papain inhibitory activity, Ca[2+]-dependent bacterial agglutination activity, robust thermostability, and specific binding capacity against multiple bacterial pathogens. These findings provide an important theoretical foundation for understanding innate immune mechanisms in P. olivaceus; however, as the current evidence is primarily based on expression profiles and in vitro assays, further in vivo studies are required to fully evaluate their potential as molecular targets for disease-resistant breeding and precise disease prevention in aquaculture.

Chemokines play important roles in coordinating innate immune defense in teleost fish. In this study, a CXCL12a homolog, designated PlCXCL12a, was cloned and characterized from leopard coral grouper (Plectropomus leopardus). PlCXCL12a encodes a conserved CXC chemokine containing a typical SCY domain and showed high sequence similarity to teleost CXCL12a homologs. PlCXCL12a was constitutively expressed in multiple tissues and was significantly induced by Singapore grouper iridovirus (SGIV), red-spotted grouper nervous necrosis virus (RGNNV), and pathogen-associated molecular pattern stimulation. Subcellular localization analysis showed that PlCXCL12a was mainly distributed in the cytoplasm and partially associated with the endoplasmic reticulum and Golgi apparatus. Functional assays demonstrated that PlCXCL12a overexpression suppressed SGIV and RGNNV replication, as evidenced by reduced viral gene expression, viral protein levels, cytopathic effects, and viral titers, while PlCXCL12a knockdown promoted viral replication. Notably, under SGIV and RGNNV co-infection, PlCXCL12a showed a stronger early inhibitory effect on SGIV but a weaker inhibitory effect on RGNNV compared with the corresponding single-infection conditions. Moreover, PlCXCL12a enhanced cell migration and increased the mRNA expression of chemokine-, inflammatory-, interferon-, and antiviral-related genes. Dual-luciferase reporter assays further showed that PlCXCL12a positively regulated NF-κB and STAT1 reporter activities. These findings suggest that PlCXCL12a functions as an inducible chemokine involved in both antiviral defense and chemotaxis-associated immune regulation in P. leopardus.

Thioredoxin (Trx) is a conserved redox regulatory protein that plays critical roles in maintaining cellular redox homeostasis and immune defense. However, its molecular characteristics and antiviral functions in grass carp (Ctenopharyngodon idella) remain largely unknown. In this study, thioredoxin 1 (trx1) was cloned and functionally characterized. The full-length trx1 cDNA was 834 bp, containing a 324 bp open reading frame encoding a 107-amino-acid protein with the conserved Cys-Gly-Pro-Cys (CGPC) redox-active motif. Phylogenetic and structural analyses demonstrated that Trx1 was highly conserved among vertebrates. The trx1 transcripts were ubiquitously expressed in all tested tissues, with relatively high levels in the spleen and liver, and were significantly induced by lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C), and grass carp reovirus (GCRV) infection. Recombinant Trx1 was successfully expressed in Escherichia coli and enhanced bacterial resistance to H2O2-induced oxidative stress. Subcellular localization analysis detected Trx1-GFP signals in the cytoplasm and nucleus of C. idella kidney (CIK) cells, with obvious nuclear enrichment after H2O2 treatment and GCRV infection. Functional analyses demonstrated that trx1 overexpression alleviated oxidative stress, inflammation, and apoptosis, accompanied by reduced GCRV replication, whereas trx1 silencing produced the opposite effects. Collectively, these findings suggest that trx1 acts as an important antiviral regulator that maintains intracellular redox homeostasis and contributes to host protection against GCRV infection by limiting oxidative stress, apoptosis, and excessive inflammatory responses.

The emergence and rapid spread of multidrug-resistant bacteria in aquaculture environments threatens the healthy development of the aquaculture industry and public health, driving demand for novel antimicrobial agents as alternatives to antibiotics. Marine animals harbor a diverse array of antimicrobial peptides, which exhibit great promise in addressing antibiotic resistance. In this work, the antimicrobial activity and mechanism of a crab-derived i-type lysozyme against multidrug-resistant Vibrio alginolyticus were investigated. The recombinant protein His-Ptlys-i (His-Ptlys-i) could bind to diverse bacteria and pathogen-associated molecular patterns (PAMPs), and displayed antibacterial and agglutinative activities against V. alginolyticus, V. parahaemolyticus, and Staphylococcus aureus. His-Ptlys-i exhibited synergistic/additive inhibitory effects against the multidrug-resistant V. alginolyticus DS016 when combined with tetracycline and fluoroquinolone antibiotics.‌ Transcriptome analysis indicated that His-Ptlys-i suppressed antibiotic resistance genes expression and inhibited multiple pathways, particularly branched-chain amino acid degradation, in V. alginolyticus DS016. Simultaneously, V. alginolyticus DS016 activates the expression of multiple ABC transporters involved in nutrient acquisition to counteract His-Ptlys-i-induced stress. His-Ptlys-i displayed promising antimicrobial activity against multidrug-resistant V. alginolyticus, indicating its potential as a candidate for novel agents combating multidrug-resistant bacteria and addressing the global challenge of antibiotic resistance.