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Background: Grass carp are an important farmed fish in China that are infected by many pathogens, especially grass carp reovirus (GCRV). Notably, grass carp showed age-dependent susceptibility to GCRV; that is, grass carp not older than one year were sensitive to GCRV, while those over three years old were resistant to this virus. However, the underlying mechanism remains unclear. Herein, whole genome-wide DNA methylation and gene expression variations between susceptible five-month-old (FMO) and resistant three-year-old (TYO) grass carp were investigated aiming to uncover potential epigenetic mechanisms. Results: Colorimetric quantification revealed that the global methylation level in TYO fish was higher than that in FMO fish. Whole-genome bisulfite sequencing (WGBS) of the two groups revealed 6214 differentially methylated regions (DMRs) and 4052 differentially methylated genes (DMGs), with most DMRs and DMGs showing hypermethylation patterns in TYO fish. Correlation analysis revealed that DNA hypomethylation in promoter regions and DNA hypermethylation in gene body regions were associated with gene expression. Enrichment analysis revealed that promoter hypo-DMGs in TYO fish were significantly enriched in typical immune response pathways, whereas gene body hyper-DMGs in TYO fish were significantly enriched in terms related to RNA transcription, biosynthesis, and energy production. RNA-seq analysis of the corresponding samples indicated that most of the genes in the above terms were upregulated in TYO fish. Moreover, gene function analysis revealed that the two genes involved in energy metabolism displayed antiviral effects. Conclusions: Collectively, these results revealed genome-wide variations in DNA methylation between grass carp of different ages. DNA methylation and gene expression variations in genes involved in immune response, biosynthesis, and energy production may contribute to age-dependent susceptibility to GCRV in grass carp. Our results provide important information for disease-resistant breeding programs for grass carp and may also benefit research on age-dependent diseases in humans. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H 2 O 2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H 2 O 2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress. • CiPrx5 and CiPrx6 enhance host resistance to H 2 O 2 and heavy metals. • Recombinant CiPrx5 and CiPrx6 proteins protect DNA against oxidative damage. • CiPrx5 and CiPrx6 inhibit GCRV replication, reduce ROS, and induce autophagy. • CiPrx5 and CiPrx6 mediate autophagy to inhibit GCRV replication and ROS. [ABSTRACT FROM AUTHOR]

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two Cu Zn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H 2 O 2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication. • Two superoxide dismutase genes were cloned and analyzed from grass carp. • Both CiSODs enhanced host resistance to H 2 O 2 and heavy metals. • Purified CiSOD proteins effectively protected DNA against oxidative damage. • CiSODs inhibit autophagy to promote GCRV replication. [ABSTRACT FROM AUTHOR]

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two Cu Zn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H 2 O 2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication. • Two superoxide dismutase genes were cloned and analyzed from grass carp. • Both CiSODs enhanced host resistance to H 2 O 2 and heavy metals. • Purified CiSOD proteins effectively protected DNA against oxidative damage. • CiSODs inhibit autophagy to promote GCRV replication. [ABSTRACT FROM AUTHOR]

Galectins, as an evolutionary conserved group of lectin superfamily, has the functions of pathogen recognition, anti-bacteria and anti-virus. In this study, a 405 bp cDNA sequence of galectin 1-like 2 (CiGal1-L2) was obtained from grass carp (Ctenopharyngodon idella), which encoded 134 amino acids with a predicted molecular mass of 15.143 kDa and an isoelectric point of 5.33. The sugar binding motifs (H–N-R, V–N and W--E-R) were detected in carbohydrate-binding domain (CRD). The amino acid sequence similarity showed that CiGal1-L2 was 40.30–42.54% and 66.42–81.20% similarity to mammalian and fish counterparts, respectively. The phylogenetic tree showed that CiGal1-L2 was clustered with fish galectin-1s and closely related to Cyprinus carpio. Real-time quantitative PCR (RT-qPCR) analysis revealed that CiGal1-L2 was widely expressed in all tested tissues. In addition, the expression of CiGal1-L2 was differentially up-regulated challenged with grass carp reovirus (GCRV), lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). The fluorescence of CiGal1-L2-GFP was distributed in the cytoplasm and nucleus of HEK 293T cells and showed a trend of nuclear translocation after LPS and poly I:C treatment. Finally, the recombinant CiGal1-L2 (rCiGal1-L2) protein showed strong binding ability to LPS. In conclusion, the results provided further insight into the immune roles of galectin-1 in teleost. • CiGal1-L2 was widely expressed in all tested tissues. • CiGal1-L2 transcripts were induced by various stimulations. • The fluorescence of CiGal1-L2-GFP showed a trend of nuclear translocation after LPS and poly I:C treatment. • The recombinant CiGal1-L2 protein showed strong binding ability to LPS. [ABSTRACT FROM AUTHOR]

Abstract Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A , RNF19B , RNF1 44AA, RNF1 44AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, in-between-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))-induced inflammatory cytokines production, including IFN-I , TNF-α , IL-6 , and transcription factor IRF3 , which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes. Highlights • Six RING-between-RING (RBR) genes from Ctenopharyngodon idellus were cloned for the first time. • RNF144AA , RNF144AB , and RNF144B responded substantially after GCRV challenge. • Poly (I: C)-induced proinflammatory cytokines were up-regulated by RNF144B silencing. • The expression of RNF144B in liver, especially in intestine was intensely up-regulated at 1 day post–infection. [ABSTRACT FROM AUTHOR]

Abstract Grass carp is an important fish species in Chinese aquaculture, and can be afflicted by a hemorrhagic disease caused by the grass carp reovirus (GCRV). Interestingly, the affects of GCRV infection of grass carp are age-restricted, meaning that one-year-old grass carp can be infected and can suffer hemorrhagic disease, but three-year-old carp are not so afflicted. In this study, we investigated the mechanism responsible for this age-restricted pathology. We evaluated the relative copy number of GCRV RNA, the expression levels of proteins in blood, and changes in DNA methylation in carp from the two age groups after infection with GCRV. After GCRV infection, the relative copy number of GCRV RNA in three-year-old grass carp was significantly lower than in one-year-old carp. The differences in circulating protein levels mainly occurred in concentrated in complement and coagulation proteins, and the expression levels of these proteins were significantly higher in three-year-old grass carp than in one-year-old carp. Moreover, the expression levels of DNA methylation-related genes in the liver and spleen of one-year-old grass carp were significantly higher than those of three-year-old carp. These results suggested that as age of grass carp increases, faster and more efficient response of the immune system after viral infection, especially the complement system, and differences in DNA methylation may be important factors that affect the age restriction observed in GCRV infection. Our study provides new insights into the mechanisms underlying age restriction of GCRV infection. Highlights • The affects of GCRV infection of grass carp are age-restricted. • The relative copy number of GCRV in three-year-old grass carp was significantly lower than in one-year-old carp. • The expression level of proteins involved in complement and blood coagulation were higher in three-year-old carp. • More efficient and faster response of the immune system may contribute to the age restriction of GCRV infection. [ABSTRACT FROM AUTHOR]

Grass carp is an important aquaculture fish species in China that is affected by severe diseases, especially haemorrhagic disease caused by grass carp reovirus (GCRV). However, the mechanisms of GCRV invasion and infection remain to be elucidated. In the present study, Ctenopharyngodon idellus kidney (CIK) cells were infected with GCRV, harvested at 0, 8, 24, and 72 h post infection, respectively, and then subjected to transcriptomics sequencing. Each sample yielded more than 6 Gb of clean data and 40 million clean reads. To better understand GCRV infection, the process was divided into three phases: the early (0–8 h post infection), middle (8–24 h post infection), and late (24–72 h) stages of infection. A total of 76 (35 up-regulated, 41 down-regulated), 553 (463 up-regulated, 90 down-regulated), and 284 (150 up-regulated, 134 down-regulated) differently expressed genes (DEGs) were identified during the early, middle, and late stages of infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that DEGs were mainly involved in carbohydrate biosynthesis, transport, and endocytosis in the early stage, phagocytosis and lysosome pathways were mainly enriched in the middle stage, and programmed cell death, apoptosis, and inflammation were largely associated with the late stage. These results suggest GCRV infection is a gradual process involving adsorption on the cell surface, followed by endocytosis into cells, transport by lysosomes, and eventually resulted in cell necrosis and/or apoptosis. Our findings provide insight into the mechanisms of grass carp reovirus infection. [ABSTRACT FROM AUTHOR]

Studies on host immunity evasion by aquatic viruses have largely focused on coding genes. There is accumulating evidence for the important biological functions of non-coding miRNAs in virus–host interactions. The regulatory functions of non-coding miRNAs in fish reovirus–host interactions remain unknown. Here, miR-2188-5p in grass carp (Ctenopharyngodon idellus), a miRNA specific to teleosts, was predicted to target the 3′ UTR of the transcription factor klf2a. A correlation analysis and dual-luciferase reporter assay revealed that miR-2188-5p could induce the degradation of klf2a. The expression of miR-2188-5p induced the degradation of klf2a in a dose-dependent manner, suppressing the type I interferon response and promoting grass carp reovirus (GCRV) replication. As determined by a co-expression analysis , klf2a inhibited viral infection when miR-2188-5p was overexpressed. The targeted degradation of klf2a by miR-2188-5p could inhibit the type I interferon response and promote the replication of GCRV; however, this targeted degradation ability was insufficient to fully inhibit GCRV infection. These results provide novel insights into the regulatory effects and biological functions of non-coding miRNAs in fish–virus interactions. • Grass carp miR-2188-5p targets the degradation of klf2a. • miR-2188-5p targets klf2a , suppresses IFN1 and promotes GCRV replication. • klf2a expression offsets the induced expression of the GCRV genome by miR-2188-5p. [ABSTRACT FROM AUTHOR]

Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region −867 ∼ +107 of noxa had high activity and the region −678 ∼ −603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPβ were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPβ were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPβ may play an important role in the regulation of noxa . Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish. [ABSTRACT FROM AUTHOR]

IPS-1, as the sole adaptor of RIG-I and MDA5, plays a central role in innate antiviral immunity. In this study, we investigated potential roles of IPS-1 in innate immunity and the domain-requirement of IPS-1 for its signaling in grass carp ( Ctenopharyngodon idella ). Overexpression experiment showed that CiIPS-1 mediated IFN-I signal possibly dependent on CiIRF7 but not CiIRF3. Post GCRV challenge, CiIPS-1 could enhance antiviral immune responses. CARD and TM domains were crucial for antiviral function of CiIPS-1, and TRAF motif played an assistant role. PRO domain seemed as a negative regulator but was pivotal for the initiation of CiIFN-I and CiMx1. Post viral/bacterial PAMPs stimulation, CiIPS-1-mediated signaling was tightly controlled. CARD domain of CiIPS-1 could significantly elicit poly I:C/LPS/PGN-mediated signaling. PRO domain negatively regulated CiIRF7 and CiIFN-I but was indispensable for inductions of CiMx1 and CiIL-1β. TRAF motif and TM domain regulated the signaling presumably in a cooperative fashion. Post poly I:C stimulation, TRAF motif negatively regulated CiIRF7, CiIFN-I and CiIL-1β at a relative early time while TM domain functioned at a relative late time. TRAF motif was indispensable for the production of CiMx1, while TM domain slightly negatively regulated the expression. Post LPS and PGN stimulation, TRAF motif excited an assistant and persistent negative role on CiIFN-I, CiIRF7 and CiIL-1β induction, but was crucial for induction of CiMx1. TM domain slightly negatively regulated LPS- and PGN-triggered signaling. Taken together, CiIPS-1 not only exerted important functions in antiviral immune response but also participated in viral/bacterial PAMPs-triggered immune response which was tightly controlled to prevent harmful effects resulting from excessive activation. This study provided novel insights into the pivotal role of IPS-1 in innate immunity. [ABSTRACT FROM AUTHOR]

Autophagy is an essential and highly conserved process in mammals, which is critical to maintaining physiological homeostasis, including cell growth, development, repair, and survival. However, the understanding of autophagy in fish virus replication is limited. In this study, we found that grass carp reovirus (GCRV) infection stimulated autophagy in the spleen of grass carp (Ctenopharyngodon idella). Moreover, both Western blot (WB) analysis and fluorescent tracer tests showed that GCRV infection induced the enhancement of autophagy activation in Ctenopharyngodon idella kidney (CIK) cells. Autophagy inducer rapamycin and autophagy inhibitor 3-MA pretreatment can inhibit and promote the proliferation of GCRV, respectively. In addition, grass carp autophagy-related gene 5 (CiATG5)-induced autophagy, as well as rapamycin, showed effects on GCRV replication in CIK cells. Transcriptome analysis revealed that the total number of differentially expressed genes (DEGs) in CiATG5 overexpression groups was less than that of the control during GCRV infection. Enrichment analysis showed that CiATG5 overexpression induced the enhancement of autophagy, lysosome, phagosome, and apoptosis in the early stage of GCRV infection, which led to the clearance of viruses. In the late stage, steroid biosynthesis, DNA replication, terpenoid backbone biosynthesis, and carbon metabolism were upregulated, which contributed to cell survival. Moreover, signaling pathways involved in the immune response and cell death were downregulated in CiATG5 overexpression groups. Further study showed that CiATG5 repressed the expression of inflammatory response genes, including cytokines and type I interferons. Taken together, the results demonstrate that autophagy represses virus replication and attenuates acute inflammatory responses to protect cells. [ABSTRACT FROM AUTHOR]

Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b−/−) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b−/− rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b−/− rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection. [ABSTRACT FROM AUTHOR]