Search

Grass carp reovirus (GCRV) causes extensive infection and death in grass carp and black carp fingerlings, with a highly seasonal prevalence. Previous studies suggested that GCRV can become latent after primary infection. In this study, we investigated type II GCRV (GCRV-II) latency in asymptomatic grass carp with GCRV infection or exposure history. We found that during latent infection, GCRV-II was detectable only in the brain of grass carp, unlike the multi-tissue distribution observed in natural infection. GCRV-II only caused damage to the brain during latent infection, while in natural infection, brain, heart, and eye tissues had relatively higher viral loads. We also discovered viral inclusion bodies in infected fish brains. Additionally, GCRV-II distribution in grass carp was notably affected by ambient temperature, with the virus targeting the brain only during low temperatures and multi-tissue distribution during high temperatures. This study provides insights into the mechanisms of GCRV-II latent infection and reactivation and contributes to the prevention and control of GCRV pandemics., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Type I IFNs with strong positive charges exhibit robust bactericidal activity and a protective effect against bacterial infections. However, the antibacterial mechanism in vivo remains unknown. In this study, Ab blockade of IFN1, a member of type I IFNs in grass carp (Ctenopharyngodon idella), resulted in high mortality, tissue bacterial loads, and low expression of immune factors after bacterial challenge, which indicates that the antibacterial activity of IFN1 has physiological significance. Meanwhile, we injected grass carp with the recombinant and purified intact IFN1 protein after bacterial injection, and the result demonstrated a remarkable therapeutic effect. Furthermore, we found that IFN1 expression was remarkably induced in blood cells after bacterial challenge, and prophagocytosis via IFN1 mostly increased in thrombocytes. Then, we isolated peripheral blood thrombocytes by polyclonal Ab of CD41 and stimulated thrombocytes with recombinant IFN1, and the results indicated that immune factors and complement components (especially C3.3) were induced. Unexpectedly, complements demonstrated not only bacteriolysis but also bacterial aggregation. Furthermore, Ab blockades of the three subunits (CRFB1/CRFB2/CRFB5) of the IFN1 receptor or inhibition of STAT1 almost abolished the prophagocytosis via IFN1 and reduced C3.3 and immune factor expression in thrombocytes. Meanwhile, Ab blockade of the complement receptor CR1 greatly attenuated the prophagocytosis of IFN1. In contrast, mouse IFN-β did not show the promotion of antibacterial activity. These results clarify the prophagocytosis and immune regulation pathways of IFN1 in antibacterial immunity in teleosts. This study reveals the antibacterial mechanisms of type I IFNs in vivo and inspires functional studies of IFN in bacterial infections., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Thrombocytes are an important component in peripheral blood cells and play a crucial role in immune regulation. CD41 is one of the biomarkers of thrombocytes. In this study, grass carp (Ctenopharyngodon idella) CD41 protein was expressed in Escherichia coli and purified by affinity chromatography. Subsequently, New Zealand rabbits were immunized with this protein via subcutaneous injection. The antibody titer examined by enzyme linked immunosorbent assay was 1:12800. The concentration of rabbit polyclonal antibody purified by HiTrap-rprotein-AFF affinity chromatography column was 1.9 mg/mL. The specificity was identified by SDS-PAGE, Western blot, flow cytometry, and indirect immunofluorescence assays. The purified antibody was used to screen grass carp thrombocytes, and CD41 + cells were 14.13%. CD41 + cells were further verified by Giemsa staining, transmission electron microscopy and RT-PCR. mRNA expression of CD41 in thrombocytes was not affected by viral or bacterial challenge in vitro, while CD41 transcripts were remarkably induced post pathogenic infections in vivo, which results from the immature hematopoietic stem cells and thrombocytes. Indirect immunofluorescence assay revealed that grass carp reovirus (GCRV) could not invade thrombocytes; however, mRNA expressions of some representative innate immune genes (IFN1, IL-1β, TNFα and Mx2) were significantly up-regulated post GCRV challenge. Meanwhile, the transcripts of some innate immune genes (IL-6 and TNFα) were swiftly increased post bacterial infection. These results indicated that the rabbit anti-CD41 polyclonal antibody possesses good specificity and can effectively bind to the CD41 protein on the surface of grass carp thrombocytes. Grass carp thrombocytes participate in immune regulation in viral and bacterial infections., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is highly infectious and lethal to grass carp, causing huge economic losses to the aquaculture industry annually. Currently, vaccination is the most effective method against viral infections. Among the various vaccination methods, the oral vaccination is an ideal way in aquaculture. However, low protective efficiency is the major problem for oral vaccination owing to some reasons, such as antigen degradation and low immunogenicity. In our study, we screened the antigenic epitopes of GCRV-II and prepared an oral microencapsulated vaccine using sodium alginate (SA) as a carrier and flagellin B (FlaB) as an adjuvant, and evaluated its protective effects against GCRV-II infection in grass carp. The full length and three potential antigenic epitope regions of GCRV-II VP56 gene were expressed in Escherichia coli and purified by glutathione affinity column respectively. The optimal antigen (VP56-3) was screened by enzyme-linked immunosorbent assay (ELISA). Adjuvant FlaB was also expressed in E. coli and purified by Ni 2+ affinity column. Subsequently, we prepared the oral vaccines using sodium alginate as a carrier. The vaccine (SA-VP56-3/FlaB) forms microsphere (1.24 ± 0.22 μm), examined by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering assay. SA-VP56-3/FlaB vaccine has excellent stability, slow-release, and low toxicity by dynamic light scattering assay, release dynamic assay, in vivo fluorescence imaging system, hemolytic activity and cytotoxicity. Then we vaccinated grass carp orally with SA-VP56-3/FlaB and measured immune-related parameters (serum neutralizing antibody titer, serum enzyme activity (TSOD, LZM, C3), immune-related genes ((IgM, IFN1, MHC-II, CD8 in head kidney and spleen), IgZ in hindgut)). The results showed that SA-VP56-3/FlaB significantly induced strong immune responses, compared to other groups. The highest survival rate achieved in SA-VP56-3/FlaB microencapsulated vaccine (56%) in 2 weeks post GCRV challenge, while 10% for the control group. Meanwhile, the tissue virus load in survival grass carp is lowest in SA-VP56-3/FlaB group. These results indicated that SA-VP56-3/FlaB could be a candidate oral vaccine against GCRV-II infection in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Qiao, Huo, Liao and Su.)

The major role of chemokines is to act as a chemoattractant to guide the migration of immune cells to the infectious sites. In the current study, we found that CiCXCL20a, a teleost-specific chemokine from grass carp ( Ctenopharyngodon idella ), demonstrates broad-spectrum, potent, direct bactericidal activity and immunomodulatory functions to bacterial infections, apart from the chemotaxis. CiCXCL20a kills bacteria by binding, mainly targeting acid lipids, perforating bacterial membrane, resulting in bacterial cytoplasm leakage and death. CiCXCL20a aggregates and neutralizes LPS, agglutinates Gram-negative bacteria, and binds to peptidoglycan and Gram-positive bacteria, but not agglutinate them. All the complexes may be phagocytized and cleared away. CiCXCL20a chemoattracts leukocytes, facilitates phagocytosis of myeloid leukocytes, not lymphoid leukocytes, and enhances the bacteria-killing ability in leukocytes. We further identified its receptor CiCXCR3.1b1. Furthermore, we investigated the physiological roles of CiCXCL20a against Aeromonas hydrophila infection in vivo. The recombinant CiCXCL20a increases the survival rate and decreases the tissue bacterial loads, edema, and lesions. Then, we verified this function by purified CiCXCL20a Ab blockade, and the survival rate decreases, and the tissue bacterial burdens increase. In addition, zebrafish ( Danio rerio ) DrCXCL20, an ortholog of CiCXCL20a, was employed to verify the bactericidal function and mechanism. The results indicated that DrCXCL20 also possesses wide-spectrum, direct bactericidal activity through membrane rupture mechanism. The present study, to our knowledge, provides the first evidence that early vertebrate chemokine prevents from bacterial infections by direct bactericidal and phagocytosis-killing-promoting manners. The results also demonstrate the close functional relationship between chemokines and antimicrobial peptides., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp ( Ctenopharyngodon idella ) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are a pivotal intracellular pattern recognition receptor family. However, studies on NLR genes in important economic fish grass carp (Ctenopharyngodon idella) are sporadic. The accumulations of genomic resource and transcriptomic sequences make it feasible to conduct a systematic analysis of these genes. In this study, we systematically conducted the genome-wide study of C. idella NLR genes and characterized their phylogeny, gene structure, conserved domain, evolutionary mechanism, and expression profiles post viral or bacterial challenge. A total of 65 NLR genes were identified and clustered into five subfamilies based on structural and phylogenetic features, including eight NODs (NLR-A), five NLRP-like receptors (NLR-B), forty-seven teleost-specific NLRs (NLR-C), two members with a B30.2 domain at the C-terminal (NLR-B30.2), and three additional NLRs (other NLRs). Gene structure analysis showed that NLRs were significantly different, with exon numbers from 3 to 31. Conserved domain analysis showed that most members of C. idella NLRs had additional domains besides the typical NLR domains. Gene duplication analysis indicated that the evolution of the NLR gene family was mainly related to segment duplication. mRNA expression analysis indicated that many members were differently expressed in multiple tissues post grass carp reovirus (GCRV) or Aeromonas hydrophila infection. The expression was particularly enhanced in liver post GCRV infection, and obviously lower post A. hydrophila infection than that post GCRV infection in spleen. These results provide systematic basic data for further functional studies of NLR, and insight into the immune responses of piscine fish NLRs to pathogen infections., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TLR22 exists in nearly all the poikilothermic vertebrates and plays a central role in the initiation of innate immunity and activation of adaptive immunity. TLR22 signaling pathway has been characterized in detail in fugu ( Takifugu rubripes ). Here, we thoroughly remold the localization and signaling pathways of TLR22. We characterized TLR22a and TLR22b in grass carp ( Ctenopharyngodon idella ), designated as CiTLR22a and CiTLR22b, and explored the ligand(s), adaptor(s), and signaling pathway(s). Results show that both CiTLR22a and CiTLR22b localize to lysosome, acidic compartment. Correspondingly, CiTLR22a and CiTLR22b directly bind and respond to dsRNA analog poly(I:C) at pH 5, but not at pH 7.4, the physiological pH. Moreover, CiTLR22a and CiTLR22b exhibit antagonistic function in signal transmission, wherein CiTLR22a facilitates the protein and phosphorylation levels of IRF7 and enhances the promoter activities of major IFNs and NF-κBs, while CiTLR22b downregulates IRF7 phosphorylation and IRF3 protein level and suppresses the IFN and NF-κB pathways. Further investigations revealed that CiTLR22a restrains grass carp reovirus (GCRV) replication and protects cells from GCRV infection, whereas CiTLR22b plays a negative role in response to GCRV infection. This is the first time to systematically clarify the signaling pathways of two isotype TLR22s; especially, subcellular localization and adaptor are different from previous TLR22 report, which results from technical limitations. The results will serve the antiviral immune mechanisms in poikilothermic vertebrates and evolutionary immunology., (Copyright © 2020 Ji, Liao, Rao, Li, Yang, Yuan, Feng, Xu, Shao and Su.)

Iron is an essential element, closely linked with host immune responses. Nevertheless, the relationship between iron metabolism and virus infection is still unclear in aquatic vertebrates. To address this issue, we employed grass carp ( Ctenopharyngodon idella ) and its lethal virus, grass carp reovirus (GCRV), a double-strand RNA virus, as models. Our results demonstrate that GCRV infection increases the iron content and alters the expression of iron metabolism-related genes both in vivo and in vitro. Of note, the expression of C. idella transferrin receptor 1 ( CiTfR1 ) rather than transferrin is upregulated upon GCRV infection. To clarify the implications of CiTfR1 upregulation for antiviral immunity, we proved that CiTfR1 was not a helper for GCRV invasion, but instead, it inhibited GCRV infection and promoted cell proliferation by facilitating the accumulation of intracellular labile iron pool (LIP), which increases intracellular oxidative stress. Interestingly, we found that CiTfR1 overexpression inhibited the mRNA expression of C. idella interferon 1 ( CiIFN1 ) and CiIFN3 . The present study reveals a novel antiviral defense mechanism in teleost where TfR1 induces the accumulation of LIP, leading to the suppression of virus infection and the proliferation of host cells, indicating that iron can be used as a medicated feed additive for the control of animal viral disease.

TANK-binding kinase 1 (TBK1) is an important kinase that regulates the activation of interferon regulatory factor 3/7 (IRF3/7) to induce type I interferon (IFN-I) production in antiviral immune responses. However, in long-term virus-host crosstalk, viruses have evolved elaborate strategies to evade host immune defense mechanisms. In the present study, we found that grass carp (Ctenopharyngodon idella) reovirus (GCRV) hijacks TBK1 to escape IRF7-IFN-Is signaling activation. In brief, GCRV inhibited TBK1 activation by restaining K63-linked ubiquitination of TBK1 and promoting its K48-linked ubiquitination. This regulation resulted in that under low titer of GCRV infection, TBK1 overexpression specifically supressed promoter activity and phosphorylation of IRF7 and induction of downstream IFN1and IFN3. qRT-PCR data uncovered that TBK1 negatively regulated IRF7, IFN1 and IFN3 transcription levels under low viral titer infection. Along with enhancement of GCRV titers, TBK1 swiched its function to up-regulate IRF7, IFN1 and IFN3 mRNA levels. Accordingly, TBK1 promoted GCRV replication at low infected titer, but inhibited GCRV replication at high infected titer. All these results revealed a viral evasion strategy that GCRV utilizes TBK1 to block cellular IFN responses at low titers or early stages in fish species, which will lay a foundation for further researching on host-virus interactions and developing novel antiviral strategies in lower vertebrates., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Complement system is an immemorial and pivotal element in innate immunity, protecting individuals from invading pathogens. Due to the emergence of whole genomes and functional researches, systematic identifications of complement system are feasible in many non-model species. In the present study, BLAST analysis was employed to systematically identify and characterize complement system in grass carp (Ctenopharyngodon idella). The results showed that C. idella complement system consists of 64 members, including the complement system pattern recognition, proteases, complement components, receptors and regulators. In which, most genes were well conserved with those in higher vertebrates over the course of evolution. Phylogenetic and syntenic analyses revealed their homologous relationships with other species. mRNA expression analyses of complement system related genes indicated that many members are sustainably expressed in multiple tissues before and after grass carp reovirus (GCRV) or Aeromonas hydrophila infection, which provide in vivo evidence for the response patterns of complement system after viral or bacterial infection. Meanwhile, this study also explored the evolution of complement system from ancestral protists to mammals and then investigated the changes in gene diversification during the evolution. These results will serve the comparative studies on the complement system in evolution and further functional investigations in C. idella., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Autophagy plays many physiological and pathophysiological roles. However, the roles and the regulatory mechanisms of autophagy in response to viral infections are poorly defined in teleost fish, such as grass carp ( Ctenopharyngodon idella ), which is one of the most important aquaculture species in China. In this study, we found that both grass carp reovirus (GCRV) infection and hydrogen peroxide (H 2 O 2 ) treatment induced the accumulation of reactive oxygen species (ROS) in C. idella kidney cells and stimulate autophagy. Suppressing ROS accumulation with N -acetyl-l-cysteine significantly inhibited GCRV-induced autophagy activation and enhanced GCRV replication. Although ROS-induced autophagy, in turn, restricted GCRV replication, further investigation revealed that the multifunctional cellular protein high-mobility group box 1b (HMGB1b) serves as a heat shock protein 70 (HSP70)-dependent, pro-autophagic protein in grass carp. Upon H 2 O 2 treatment, cytoplasmic HSP70 translocated to the nucleus, where it interacted with HMGB1b and promoted cytoplasmic translocation of HMGB1b. Overexpression and siRNA-mediated knockdown assays indicated that HSP70 and HMGB1b synergistically enhance ROS-induced autophagic activation in the cytoplasm. Moreover, HSP70 reinforced an association of HMGB1b with the C. idella ortholog of Beclin 1 (a mammalian ortholog of the autophagy-associated yeast protein ATG6) by directly interacting with C. idella Beclin 1. In summary, this study highlights the antiviral function of ROS-induced autophagy in response to GCRV infection and reveals the positive role of HSP70 in HMGB1b-mediated autophagy initiation in teleost fish., (© 2018 Rao et al.)

Grass carp (Ctenopharyngodon idella) is an important economic species in freshwater aquaculture and its industry has been confined due to variety degeneration and frequent diseases. Marker-assisted selection is a feasible method for selective breeding of new varieties. Transcriptome data have greatly facilitated high-throughput single nucleotide polymorphism (SNP) marker discovery and phenotype association study. In this study, we gained a total of 25,981 and 5,775 high quality SNPs in two transcriptomes from individuals and cell lines, respectively. Comparative transcriptome analysis identified 413 and 832 grass carp reovirus (GCRV)-resistant-association SNPs as well as 1,381 and 1,606 GCRV-susceptible-association SNPs in individuals and cell lines, respectively. Integrated analysis indicated 22 genes with single SNP share common resistant/susceptible traits in two transcriptomes. Furthermore, we infected grass carp with GCRV, genotyping and association analyses were performed, and 9 in 22 SNPs were confirmed by PCR-RFLP. Meanwhile, mRNA expression profiles of 6 genes containing confirmed SNPs were examined by qRT-PCR. The results demonstrated that mRNA expressions were significant differences in resistant/susceptible individuals and cell lines. The present study develops an important strategy for high throughput screening of phenotype association genetic markers and the results will serve in grass carp breeding for GCRV resistance.

Interferons (IFNs) play crucial roles in the immune response of defense against viral infection and bacteria invasion. In the present study, we systematically identified and characterized the IFNs, their regulatory factors (Interferon Regulatory Factors, IRFs) and receptors (Cytokine Receptor Family B, CRFBs) in grass carp (Ctenopharyngodon idella). Grass carp IFNs can be classified into type I IFN (IFN-I) and type II IFN (IFN-II) like other teleosts. IFN-I consist of two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into three subgroups (IFN-a, -c and -d), containing four members: IFN1, IFN2, IFN3, IFN4 in grass carp. IFN-II contain two members, IFNγ2 with the similarity to mammalian IFNγ and a cyprinid specific IFNγ1 (IFNγ-rel) molecule. mRNA expression analyses of IFNs discovered that IFN1 and IFN-II were sustainably expressed in many tissues, while other IFN members were transiently expressed in specific tissues and time points. In the immune response, IFN transcriptions are primarily regulated through multiple IRFs after grass carp reovirus (GCRV) challenge. IRF family possess thirteen members in grass carp, which can be further divided into four subfamilies (IRF-1, -3, -4 and -5 subfamily), each of them plays different roles in the innate and adaptive immunity via various signaling pathways to interact with IFNs (mainly IFN-I). IFNs have to bind receptors (CRFBs) to perform their functions. CRFBs as IFN receptors contain six members in grass carp. The structure and expression characterizations of IFNs, IRFs and CRFBs were analyzed using bioinformatics tools. These results might provide basic data for the further functional research of IFN system, and deeply understand fish immune mechanisms against virus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)