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Peroxiredoxins are a family of antioxidant proteins that protect cells from oxidative damage caused by reactive oxygen species (ROS). Herein, the peroxiredoxin 3 gene from grass carp (Ctenopharyngodon idellus), named CiPrx3, was cloned and analyzed. The full-length cDNA of CiPrx3 is 1068 bp long, with a 753 bp open reading frame (ORF) that contains a thioredoxin-2 domain, two peroxiredoxin signature motifs, and two highly conserved cysteine residues. CiPrx3 was ubiquitously expressed in all the tested tissues, while its expression level was altered significantly after exposure to grass carp reovirus (GCRV) and pathogen-associated molecular patterns (PAMPs). CiPrx3 was localized in the mitochondria of transfected cells and concentrated in the nucleus after poly (I:C) treatment. Transformation of CiPrx3 into Escherichia coli enhanced host resistance to H2O2 and heavy metals. Purified recombinant CiPrx3 proteins could protect DNA against oxidative damage. Overexpression of CiPrx3 in fish cells reduced intracellular ROS, increased cell viability, and decreased cell apoptosis caused by H2O2 stimulation and GCRV infection. Further study indicated that CiPrx3 induced autophagy to inhibit GCRV replication in fish cells. Collectively, these results imply that grass carp Prx3 elevates host antioxidant activity and induces autophagy to inhibit GCRV replication. [ABSTRACT FROM AUTHOR]

Grass carp (Ctenopharyngodon idellus) is an important aquaculture species in China that is affected by serious diseases, especially hemorrhagic disease caused by grass carp reovirus (GCRV). Grass carp have previously shown age-dependent susceptibility to GCRV, however, the mechanism by which this occurs remains poorly understood. Therefore, we performed transcriptome and metabolome sequencing on five-month-old (FMO) and three-year-old (TYO) grass carp to identify the potential mechanism. Viral challenge experiments showed that FMO fish were susceptible, whereas TYO fish were resistant to GCRV. RNA-seq showed that the genes involved in immune response, antigen presentation, and phagocytosis were significantly upregulated in TYO fish before the GCRV infection and at the early stage of infection. Metabolome sequencing showed that most metabolites were upregulated in TYO fish and downregulated in FMO fish after virus infection. Intragroup analysis showed that arachidonic acid metabolism was the most significantly upregulated pathway in TYO fish, whereas choline metabolism in cancer and glycerophospholispid metabolism were significantly downregulated in FMO fish after virus infection. Intergroup comparison revealed that metabolites from carbohydrate, amino acid, glycerophospholipid, and nucleotide metabolism were upregulated in TYO fish when compared with FMO fish. Moreover, the significantly differentially expressed metabolites showed antiviral effects both in vivo and in vitro. Based on these results, we concluded that the immune system and host biosynthesis and metabolism, can explain the age-dependent viral susceptibility in grass carp. [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]

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]

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]

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]

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]

Abstract: The mammalian response to lipopolysaccharide (LPS) is mainly mediated by Toll-Like Receptor 4 (TLR4). Fish and mammalian TLR4 vary; fish TLR4 ligands are unknown. Isolation of fish tlr4 genes is difficult due to their complex genomic structure. Three bacterial artificial chromosome (BAC) clones containing grass carp tlr4 were obtained. Four tlr4 genes, with a varied genomic structure and different protein domains were subsequently isolated by constructing a subcloned library and rapid amplification of cDNA ends (RACE). The four tlr4 genes were expressed during development from 12h post-fertilization, in all healthy adult fish tissues tested, and significantly increased in grass carp reovirus (GCRV)-infected liver and muscle, suggesting the tlr4 genes play a role in GCRV infection. This study effectively separated each gene in the tlr4 gene cluster, implies that grass carp TLR4 proteins have different ligand recognition specificities to mammalian TLRs, and provides information on the functional evolution of TLRs. [Copyright &y& Elsevier]