Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Comparative Transcriptomics Analysis Reveals Unique Immune Response to Grass Carp Reovirus Infection in Barbel Chub (Squaliobarbus curriculus).

Grass carp (Ctenopharyngodon idella) and barbel chub (Squaliobarbus curriculus)-both Leuciscinae subfamily species-demonstrate differences in grass carp reovirus (GCRV) infection resistance. We infected barbel chubs with type II GCRV and subjected their liver, spleen, head kidney, and trunk kidney samples to investigate anti-GCRV immune mechanisms via RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). We identified 139, 970, 867, and 2374 differentially expressed genes (DEGs) in the liver, spleen, head kidney, and trunk kidney, respectively. Across all four tissues, gene ontology analysis revealed significant immune response-related DEG enrichment, and the Kyoto Encyclopedia of Genes and Genomes analysis revealed pattern recognition receptor (PRR) and cytokine-related pathway enrichment. We noted autophagy pathway enrichment in the spleen, head kidney, and trunk kidney; apoptosis pathway enrichment in the spleen and trunk kidney; and complement- and coagulation-cascade pathway enrichment in only the spleen. Comparative transcriptome analysis between GCRV-infected barbel chubs and uninfected barbel chubs comprehensively revealed that PRR, cytokine-related, complement- and coagulation-cascade, apoptosis, and autophagy pathways are potential key factors influencing barbel chub resistance to GCRV infection. qRT-PCR validation of 11 immune-related DEGs confirmed our RNA-seq data's accuracy. These findings provide a theoretical foundation and empirical evidence for the understanding of GCRV infection resistance in barbel chub and hybrid grass carp-barbel chub breeding.

Identification of the C1qDC gene family in grass carp (Ctenopharyngodon idellus) and the response of C1qA, C1qB, and C1qC to GCRV infection in vivo and in vitro.

Proteins from the C1q domain-containing (C1qDC) family recognize self-, non-self-, and altered-self ligands and serves as an initiator molecule for the classical complement pathway as well as recognizing immune complexes. In this study, C1qDC gene family members were identified and analyzed in grass carp (Ctenopharyngodon idellus). Members of the C1q subfamily were cloned, and their response to infection with the grass carp virus was investigated. In the grass carp genome, 54 C1qDC genes and 67 isoforms have been identified. Most were located on chromosome 3, with 52 shared zebrafish homologies. Seven substantially differentially expressed C1qDC family genes were identified in the transcriptomes of cytokine-induced killer (CIK) cells infected with grass carp reovirus (GCRV), all of which exhibited sustained upregulation. The opening reading frames of grass carp C1qA, C1qB, and C1qC, belonging to the C1q subfamily, were determined to be 738, 732, and 735 base pairs, encoding 245, 243, and 244 amino acids with molecular weights of 25.81 kDa, 25.63 kDa and 26.16 kDa, respectively. Three genes were detected in the nine collected tissues, and their expression patterns were similar, with the highest expression levels observed in the spleen. In vivo after GCRV infection showed expression trends of C1qA, C1qB, and C1qC in the liver, spleen, and kidney. An N-type pattern in the liver and kidney was characterized by an initial increase followed by a decrease, with the highest expression occurring during the recovering period, and a V-type pattern in the spleen with the lowest expression levels during the death period. In vitro, after GCRV infection showed expression trends of C1qA, C1qB, and C1qC, and this gradually increased within the first 24 h, with a notable increase observed at the 24 h time point. After CIK cells incubation with purified recombinant proteins, rC1qA, rC1qB, and rC1qC for 3 h, followed by GCRV inoculation, the GCRV replication indicated that rC1qC exerted a substantial inhibitory effect on viral replication in CIK cells after 24 h of GCRV inoculation. These findings offer valuable insights into the structure, evolution, and function of the C1qDC family genes and provide a foundational understanding of the immune function of C1q in grass carp.

The Teleost CXCL13-CXCR5 Axis Induces Inflammatory Cytokine Expression through the Akt-NF-κB, p38-AP-1, and p38-NF-κB Pathways.

The ancestors of chemokines originate in the most primitive of vertebrates, which has recently attracted great interest in the immune functions and the underlying mechanisms of fish chemokines. In the current study, we identified an evolutionarily conserved chemokine, CiCXCL13, from a teleost fish, grass carp. CiCXCL13 was characterized by a typical SCY (small cytokine CXC) domain and four cysteine residues (C34, C36, C61, C77), with the first two cysteines separated by a random amino acid residue, although it shared 24.2-54.8% identity with the counterparts from other vertebrates. CiCXCL13 was an inducible chemokine, whose expression was significantly upregulated in the immune tissues of grass carps after grass carp reovirus infection. CiCXCL13 could bind to the membrane of grass carp head kidney leukocytes and promote cell migration, NO release, and the expression of >15 inflammatory cytokines, including IL-1ß, TNF-α, IL-10 and TGF-ß1, thus regulating the inflammatory response. Mechanistically, CiCXCL13 interacted with its evolutionarily conserved receptor CiCXCR5 and activated the Akt-NF-κB and p38-AP-1 pathways, as well as a previously unrevealed p38-NF-κB pathway, to efficiently induce inflammatory cytokine expression, which was distinct from that reported in mammals. Zebrafish CXCL13 induced inflammatory cytokine expression through Akt, p38, NF-κB, and AP-1 as CiCXCL13. Meanwhile, the CiCXCL13-CiCXCR5 axis-mediated inflammatory activity was negatively shaped by grass carp atypical chemokine receptor 2 (CiACKR2). The present study is, to our knowledge, the first to comprehensively define the immune function of CXCL13 in inflammatory regulation and the underlying mechanism in teleosts, and it provides a valuable perspective on the evolution and biology of fish chemokines.

TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.

First in vitro and in vivo evaluation of recombinant IL-1ß protein as a potential immunomodulator against viral infection in fish.

IL-1ß is an important proinflammatory cytokine with multifaceted modulatory roles in immune responses. In fish, recombinant IL-1ß has been employed in the control of bacterial diseases, while the antiviral mechanisms of IL-1ß remain largely unknown, and the efficacy of recombinant IL-1ß as an immunomodulator to prevent viral diseases is still not determined. This study evaluated the immunomodulatory effects of recombinant grass carp IL-1ß against grass carp reovirus (GCRV) in vitro and in vivo. Firstly, the mature form (Ser111-Lys270) of grass carp IL-1ß was identified, and its recombinant protein (designated as rgcIL-1ß) was prepared through prokaryotic expression. Then, an in vitro evaluation model for rgcIL-1ß activity was established in the CIK cells, with the appropriate concentration (600 ng/mL) and effect time (1 h). In vitro, rgcIL-1ß could not only induce the production of proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α but also a series of antiviral factors including IFN-1, IFN-2, IFN-γ, and ISG15. Mechanistically, transcriptome analysis and western blotting confirmed that rgcIL-1ß activated multiple transcriptional factors, including NF-κB, IRF1, IRF3, and IRF8, and the signal pathways associated with inflammatory cytokines and antiviral factors expression. Expectedly, rgcIL-1ß treatment significantly inhibited GCRV replication in vitro. In vivo administration of rgcIL-1ß via intraperitoneal pre-injection significantly aroused an antiviral response to restrict GCRV replication and intense tissue inflammation in grass carp, demonstrating the immunomodulatory effects of rgcIL-1ß. More importantly, rgcIL-1ß administrated with 10 ng/g and 1 ng/g could improve the survival rate of grass carp during GCRV infection. This study represents the first time to comprehensively reveal the immunomodulatory and antiviral mechanisms of IL-1ß in fish and may also pave the way for further developing recombinant IL-1ß as an immunotherapy for the prevention and control of fish viral diseases.

JunD functions as a transcription factor of IL-10 to regulate bacterial infectious inflammation in grass carp (Ctenopharyngodon idella).

IL-10 is a key anti-inflammatory mediator ensuring the protection of a host from excessive inflammation in response to pathogen infections, whose transcription or expression levels are tightly linked to the onset and progression of infectious diseases. An AP-1 family member called CiJunD was shown to be a transcription factor of IL-10 in grass carp (Ctenopharyngodon idella) in the current study. CiJunD protein harbored the conserved Jun and bZIP domains. Mutant experiments demonstrated that CiJunD bound to three specific sites on IL-10 promoter, i.e., 5'-ATTATTCATA-3', 5'-AGATGAGACATCT-3', and 5'-ATTATTCATC-3', mainly relying on the bZIP domain, and initiated IL-10 transcription. Expression data from the grass carp spleen infected by Aeromonas hydrophila and lipopolysaccharide (LPS) challenged spleen leukocytes indicated that the expressions of CiJunD and IL-10 were positively correlated, while the expression of pro-inflammatory cytokines, such as IL-1ß, IL-6, IL-8, IFN-γ, and TNF-α, showed an overall downward trend when CiJunD and IL-10 peaked. The ability of CiJunD to down-regulate the production of pro-inflammatory cytokines and up-regulate the expression of IL-10, both with and without LPS stimulation, was confirmed by overexpression experiments. Meanwhile, the subcellular fractionation assay revealed that the nuclear translocation of CiJunD was significantly enhanced after the LPS challenge. Moreover, in vivo administration of grass carp with Oxamflatin, a potent agonist of JunD activity, could promote IL-10 but suppress the expression of pro-inflammatory cytokines. Intriguingly, tissue inflammation lesions and the survival rates of grass carp infected with A. hydrophila were also significantly improved by Oxamflatin administration. This work sheds light on the regulation mechanism by JunD of IL-10 expression and bacterial infectious inflammation for the first time, and it may present a viable method for preventing infectious diseases in fish by regulating IL-10 expression and inflammatory response.

Structural comparison and expression function analysis of BF/C2 in Ctenopharyngodon idella and Squaliobarbus curriculus.

Ctenopharyngodon idella and Squaliobarbus curriculus, members of the Cyprinidae family and Yaroideae subfamily, have shown different levels of resistance to grass carp reo virus (GCRV), with S. curriculus exhibiting higher resilience. In the pursuit to explore the distinctions in the structural and expression traits of BF/C2 (A,B) between the two species, we conducted an analysis involving the cloning and examination of various coding sequences (CDS). We successfully cloned the CDS of ci-BF/C2A and ci-BF/C2B from C. idella, which spanned 2259 bp and 2514 bp respectively, encoding 752 and 837 amino acids. Similarly, the CDS of sc-BF/C2A and sc-BF/C2B from S. curriculus were cloned, featuring lengths of 1353 bp and 2517 bp and encoding 450 and 838 amino acids, respectively. A chromosome collinearity assessment revealed that ci-BF/C2A demonstrated collinearity with sc-BF/C2A, a finding not replicated with ci-BF/C2B and sc-BF/C2B. Delving into gene structure, we discerned that ci-BF/C2A harbored a greater number of Tryp_SPc domains compared to sc-BF/C2A. Following this, we engineered and purified six prokaryotic recombinant proteins: CI-BF/C2A, CI-BF/C2A1 (a variant resulting from the deletion of the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2A2 (representing the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2B, SC-BF/C2A, and SC-BF/C2B. Through serum co-incubation tests with these recombinant proteins, we established the activation of the complement marker C3 in each case. Utilizing fluorescence quantitative expression analysis, we observed ubiquitous expression of ci-BF/C2A and ci-BF/C2B across all grass carp tissues, predominantly in the liver. This pattern mirrored in S. curriculus, where sc-BF/C2A was highly expressed in the gills, and sc-BF/C2B manifested notably in the liver. Kidney cell infection experiments on both species revealed enhanced resistance to GCRV post-incubation with the recombinant proteins. Notably, cells treated with SC-BF/C2 (A, B) exhibited pronounced resilience compared to those treated with CI-BF/C2 (A, B, A1, A2). However, cells incubated with CI-BF/C2A1 and CI-BF/C2A2 showed strengthen resistance relative to cells treated with CI-BF/C2A and CI-BF/C2B. In GCRV infection trials on grass carp, ci-BF/C2A and ci-BF/C2B expressions reached a zenith on the seventh day post-infection, highlighting a distinctive functional mode in immune defense against GCRV infection orchestrated by BF/C2. The empirical data underscores the pivotal role of the Tryp_SPc domain in immune responses to GCRV infection, pinpointing its influence on ci-BF/C2A expression. Conclusively, this investigation provides a foundational understanding of the unique immune function characteristics of BF/C2 in grass carp, paving the way for further scholarly exploration in this realm.

Nucleophosmin 1a translocated from nucleus to cytoplasm and facilitate GCRV replication.

To decipher the functional characterization of Nucleophosmin 1a (NPM1a) from grass carp (Ctenopharyngodon idellus) (CiNPM1a), its cDNA was cloned and bioinformatic analysis were conducted. The full-length cDNA sequence of CiNPM1a is 1732 bp, which encodes 307 amino acids. CiNPM1a contains conserved domains of Nucleoplasmin domain, NPM1-C terminal domain, as well as nuclear localization signals, nuclear export signal (NES) and acid patches. There are 52 and 20 consensus amino acids exist in the Nucleoplasmin domain and the NPM1-C terminal domain of all blasted species. In addition, the immune function of CiNPM1a were analyzed. The Ciirf7, Ciifn1 and Ciifn2 transcription was inhibited, whereas the vp2 and vp7 expressions were enhanced in CiNPM1a overexpressing cells after GCRV infection (P < 0.05). Moreover, the Ciirf7, Ciifn1 and Ciifn2 mRNA levels were significantly up-regulated, but the vp2 and vp7 expressions were significantly down-regulated in CiNPM1a knockdown cells after infection. This indicated that CiNPM1a played negative roles in the induction of Type I IFN reaction and thus the GCRV replication. Finally, the NES domain that affect the nucleous-cytoplasm shuttle and the replication of GCRV were investigated. The deletion of NES1 and NES(1 + 2+3) absolutely limited the transloacation of CiNPM1a△NES1 protein and CiNPM1a △NES(1 + 2+3) protein to cytoplasm after infection, and the deletion of NES2 resulted in partially limitation of protein shuttle. In general, Ciirf3, Ciirf7, Ciifn1 and Ciifn2 expressions were enhanced in the CiNPM1a△NES1, CiNPM1a△NES2 and CiNPM1a△NES3 overexpression groups, and the deletion of functional domains in CiNPM1a led to significantly reduction of the vp2 and vp7 replication. The results indicated that CiNPM1a may be a target molecular for GCRV infection curation, and a candidate molecular for resistance strain breeding of grass carp.

Correction: An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

[This corrects the article DOI: 10.1371/journal.ppat.1011320.].

An Aquareovirus Exploits Membrane-Anchored HSP70 To Promote Viral Entry.

Temperature dependency of viral diseases in ectotherms has been an important scientific issue for decades, while the molecular mechanism behind this phenomenon remains largely mysterious. In this study, deploying infection with grass carp reovirus (GCRV), a double-stranded RNA aquareovirus, as a model system, we demonstrated that the cross talk between HSP70 and outer capsid protein VP7 of GCRV determines temperature-dependent viral entry. Multitranscriptomic analysis identified HSP70 as a key player in the temperature-dependent pathogenesis of GCRV infection. Further biochemical, small interfering RNA (siRNA) knockdown, pharmacological inhibition, and microscopic approaches revealed that the primary plasma membrane-anchored HSP70 interacts with VP7 to facilitate viral entry during the early phase of GCRV infection. Moreover, VP7 functions as a key coordinator protein to interact with multiple housekeeping proteins and regulate receptor gene expression, concomitantly facilitating viral entry. This work illuminates a previously unidentified immune evasion mechanism by which an aquatic virus hijacks heat shock response-related proteins to enhance viral entry, pinpointing targeted preventives and therapeutics for aquatic viral diseases. IMPORTANCE The seasonality of viral diseases in ectotherms is a prevailing phenomenon in the aquatic environment, which causes huge economic losses every year worldwide and hinders sustainable development of the aquaculture industry. Nevertheless, our understanding of the molecular mechanism of how temperature determines the pathogenesis of aquatic viruses remains largely unexplored. In this study, by deploying grass carp reovirus (GCRV) infection as a model system, we demonstrated that temperature-dependent, primarily membrane-localized HSP70 interacts with major outer capsid protein VP7 of GCRV to bridge the virus-host interaction, reshape the host's behaviors, and concomitantly facilitate viral entry. Our work unveils a central role of HSP70 in the temperature-dependent pathogenesis of aquatic viruses and provides a theoretical basis for the formulation of prevention and control strategies for aquatic viral diseases.

Astragalus polysaccharide improves the growth, meat quality, antioxidant capacity and bacterial resistance of Furong crucian carp (Furong carp♀ × red crucian carp♂).

To evaluate the functional effects of APS (Astragalus polysaccharide) on Furong crucian carp, APS-supplemented diets (0.00 %, 0.05 %, 0.10 % and 0.15 %) were prepared and utilized in feeding experiment. The results showed that the 0.05 % APS group has the highest weight gain rate and specific growth rate, and the lowest feed coefficient rate. In addition, 0.05 % APS supplement could improve muscle elasticity, adhesiveness and chewiness. Moreover, the 0.15 % APS group had the highest spleen-somatic index and the 0.05 % group had the maximum intestinal villus length. 0.05 % and 0.10 % APS addition significantly increased T-AOC and CAT activities while MDA contents decreased in all APS groups. The plasma TNF-α levels in all APS groups significantly increased (P<0.05), and the 0.05 % group showed the highest TNF-α level in spleen. In APS addition groups, the tlr8, lgp2 and mda5 gene expressions were significantly elevated, while xbp1, caspase-2 and caspase-9 expressions decreased in uninfected and A. hydrophila-infected fish. Finally, higher survival rate and slower disease outbreak rate were observed in APS-supplemented groups after being infected by A. hydrophila. In conclusion, Furong crucian carp fed by APS-supplemented diets possesses elevated weight gain rate and specific growth rate, and improved meat quality, immunity and disease resistance.

An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases.

Zebrafish MARCH7 negatively regulates IFN antiviral response by degrading TBK1.

Membrane-associated RING-CH-type finger (MARCH) proteins have been reported to regulate type I IFN production during host antiviral innate immunity. The present study reported the zebrafish MARCH family member, MARCH7, as a negative regulator in virus-triggered type I IFN induction via targeting TANK-binding kinase 1 (TBK1) for degradation. As an IFN-stimulated gene (ISG), we discovered that MARCH7 was significantly induced by spring viremia of carp virus (SVCV) or poly(I:C) stimulation. Ectopic expression of MARCH7 reduced the activity of IFN promoter and dampened the cellular antiviral responses triggered by SVCV and grass carp reovirus (GCRV), which concomitantly accelerated the viral replication. Accordingly, the knockdown of MARCH7 by siRNA transfection significantly promoted the transcription of ISG genes and inhibited SVCV replication. Mechanistically, we found that MARCH7 interacted with TBK1 and degraded it via K48-linked ubiquitination. Further characterization of truncated mutants of MARCH7 and TBK1 confirmed that the C-terminal RING of MARCH7 is essential in the MARCH7-mediated degradation of TBK1 and the negative regulation of IFN antiviral response. This study reveals a molecular mechanism by which zebrafish MARCH7 negatively regulates the IFN response by targeting TBK1 for protein degradation, providing new insights into the essential role of MARCH7 in antiviral innate immunity.

Identification and expression analysis of zebrafish gnaq in the hypothalamic-Pituitary-Gonadal axis.

The G proteins have emerged as essential molecular switches in a wide variety of signal transduction pathways. Gαq, encoded by G protein subunit alpha q (gnaq), is a member of the G proteins and participates in regulating important biological activities in mammals; however, its function and regulatory mechanism in teleost remain largely unclear. In the current study, we cloned the cDNA of gnaq from zebrafish (Danio rerio) and investigated the expression characteristics of Gαq/gnaq in reproductive tissues. RT-PCR and WISH analyses showed that gnaq was widely expressed in zebrafish tissues, with high expression in the brain, olfactory brain, and hypothalamus. During the embryonic development stage, the gnaq was mainly distributed in the hypothalamus after 72 h post-fertilization. In addition, immunohistochemistry analysis revealed that the Gαq protein was highly expressed in the diffuse nucleus of the inferior hypothalamic lobe (DIL), ventral zone of the periventricular hypothalamus (Hv), and caudal zone of the periventricular hypothalamus (Hc) in adult zebrafish. Furthermore, in the gonads, the Gαq protein was found in oocytes of all stages, except spermatids. Lastly, the gnaq mRNA exhibited relatively low expression in gonads on Day 4 during the reproductive cycle, while increasing drastically in the hypothalamus and pituitary afterward. Altogether, our results suggest that gnaq/Gαq might be important in fish reproduction.

Functional and Expressional Analyses Reveal the Distinct Role of Complement Factor I in Regulating Complement System Activation during GCRV Infection in Ctenopharyngodon idella.

Complement factor I (CFI), a complement inhibitor, is well known for regulating the complement system activation by degrading complement component 3b (C3b) in animal serum, thus becoming involved in innate defense. Nevertheless, the functional mechanisms of CFI in the complement system and in host-pathogen interactions are far from being clarified in teleost fish. In the present study, we cloned and characterized the CFI gene, CiCFI, from grass carp (Ctenopharyngodon idella) and analyzed its function in degrading serum C3b and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiCFI was found to be 2121 bp, encoding 706 amino acids with a molecular mass of 79.06 kDa. The pairwise alignments showed that CiCFI shared the highest identity (66.9%) with CFI from Carassius gibelio and the highest similarity (78.7%) with CFI from Danio rerio. The CiCFI protein was characterized by a conserved functional core Tryp_SPc domain with the catalytic triad and substrate binding sites. Phylogenetic analysis indicated that CiCFI and the homologs CFIs from other teleost fish formed a distinct evolutionary branch. Similar with the CFIs reported in mammals, the recombinant CiCFI protein could significantly reduce the C3b content in the serum, demonstrating the conserved function of CiCFI in the complement system in the grass carp. CiCFI mRNA and protein showed the highest expression level in the liver. After GCRV infection, the mRNA expressions of CiCFI were first down-regulated, then up-regulated, and then down-regulated to the initial level, while the protein expression levels maintained an overall downward trend to the late stage of infection in the liver of grass carps. Unexpectedly, the protein levels of CiCFI were also continuously down-regulated in the serum of grass carps during GCRV infection, while the content of serum C3b proteins first increases and then returns to the initial level, suggesting a distinct role of CiCFI in regulating complement activation and fish-virus interaction. Combining our previous results that complement factor D, a complement enhancer, shows continuously up-regulated expression levels in grass carps during GCRV infection, and this study may provide the further essential data for the full picture of complex complement regulation mechanism mediated by Df and CFI of the grass carp during pathogen infection.

Effect of dietary replacement of fish meal by poultry by-product meal on the growth performance, immunity, and intestinal health of juvenile red swamp crayfish, procambarus clarkia.

The present study was conducted to investigate the dietary replacement of fish meal with poultry by-product meal (PBM) on the growth performance, immunity, antioxidant properties, and intestinal health of red swamp crayfish (Procambarus clarkia). A diet containing 20% fish meal (FM) and complex plant ingredients as the main protein resources was set as the FM group (crude protein 32%, crude lipid 6%). Four diets replacing 25%, 50%, 75%, and 100% fish meal of the FM diet with PBM were set as the PBM25, PBM50, PBM75, and PBM100 groups, respectively. Compared to the FM group, the PBM100 diet significantly decreased growth performance and feed utilization of crayfish, while markedly increasing the activity of serum aspartate aminotransferase. The immune response was depressed in crayfish fed the PBM100 diet as the activities of serum lysozyme and phenoloxidase, gene expression of anti-lipopolysaccharide factors (alf), cyclophilin A (cypa), crustin, and hemocyanin-1 (hep-1) in hepatopancreas were remarkably decreased. The activities of antioxidases and expression of antioxidant-relevant genes in the hepatopancreas were not influenced by PBM inclusion. Crayfish fed different diets exhibited no obvious symptoms of enteritis, but the PBM100 diet destructed intestinal morphology by significantly decreasing the average length of longitudinal ridges. The α-diversity and overall community structure were not significantly influenced but variations were found in the relative abundance of some genera by PBM inclusion. In summary, CAP could successfully replace 75% dietary FM in a basal diet containing 20% fish meal, while higher CAP level compromised growth performance, immunity, and intestinal histology of crayfish.

Functional and expression analysis reveals the involvement of integrin αvß3 in antiviral immunity of grass carp (Ctenopharyngodon idella).

Integrins are α-ß heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvß3 (GCαvß3) originally assembled by αv (GCαv) and ß3 (GCß3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCß3 shared high similarity (75.2-95.1%) and identity (58.6-90.7%) with their homologs from other vertebrates. Both GCαv and GCß3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCß3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCß3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvß3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvß3 did. In addition, both GCαv and GCß3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvß3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.

Sequence, Expression, and Anti-GCRV Function of the Ferritin from the Grass Carp, Ctenopharyngodon idellus.

Ferritin possesses an immune function to defend against pathogen infection. To elucidate the immunity-protecting roles of ferritin from Ctenopharyngodon idellus (Ciferritin) against virus infection, the cDNA and promoter sequences of Ciferritin were determined, and the correlations between Ciferrtin expressions and promoter methylation levels were analyzed. In addition, the functional role of Ciferrtin on GCRV (grass carp reovirus) infection was assessed. The full-length cDNA of Ciferritin is 1053 bp, consists of a 531 bp open-reading frame, and encodes 176 amino acids. Ciferritin showed the highest sequence identity with the ferritin middle subunit of Mylopharyngodon piceus (93.56%), followed by the subunits of Megalobrama amblycephala and Sinocyclocheilus rhinocerous. Ciferritin contains a conserved ferritin domain (interval: 10−94 aa), and the caspase recruitment domain (CARD) and Rubrerythrin domain were also predicted. In the spleen and kidney, significantly higher Ciferritin expressions were observed at 6, 12, 24, or 168 h post GCRV infection than those in the PBS injection group (p < 0.05). The Ciferrtin expression level in the progeny of maternal-immunized grass carp was significantly higher than that in the progeny of common grass carp (p < 0.05). Ciferritin promoter methylation level in the progeny from common grass carp was 1.27 ± 0.15, and in the progeny of the maternal-immunized group was 1.00 ± 0.14. In addition, methylation levels of "CpG9" and "CpG10" loci were significantly lower in the progeny of maternal-immunized fish than those in the common group. Except for the "CpG5", methylation levels of all other detected "CpG" loci negatively correlated with Ciferritin expression levels. Furthermore, the total methylation level of "CpG1−10" negatively correlated with the Ciferritin expressions. The Ciferritin expression level was significantly up-regulated, and the VP7 protein levels were significantly reduced, at 24 h post GCRV infection in the Ciferritin over-expression cells (p < 0.05). The results from the present study provide sequence, epigenetic modification and expression, and anti-GCRV functional information of Ciferritin, which provide a basis for achieving resistance to GCRV in grass carp breeding.