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Rab GTPases not only play a crucial role in the exocytosis and membrane transport of all eukaryotic cells, but are also gradually being found to be involved in innate immunity in mammals. However, the immunological function of Rab3a in fish remains unclear. In this study, two members of Rab3a, CiRab3aa and CiRab3ab, were cloned from grass carp (Ctenopharyngodon idella) for the first time. It was observed that CiRab3aa exhibited a prompt response to grass carp reovirus (GCRV) infection, and suppressing the expression of the CiRab3aa effectively inhibited GCRV replication, whereas the expression level of the CiRab3ab gene did not affect GCRV replication. Furthermore, overexpression of CiRab3aa significantly suppressed the mRNA levels of TLR9 , TNF-α , IL-1β , IL-6 , IL-10 , ERK , and IFN-I following stimulation with CpG ODN 1670 A, suggesting that CiRab3aa is a negative regulator of TLR9 signaling pathway. In conclusion, this study not only revealed functional differences between CiRab3aa and CiRab3ab in grass carp immunity, providing new sights into understanding their function in teleost fish, but also provided a new target for the treatment strategy of grass carp hemorrhagic disease. These results will help address the harm and economic losses caused by GCRV infection in grass carp breeding industry. • CiRab3aa and CiRab3ab genes were cloned and characterized in grass carp. • There are 7 amino acid differences in the Rab domain of Rab3aa and Rab3aa protein. • Knockdown of CiRab3aa significantly inhibited the replication of GCRV. • CiRab3aa was a negative regulator of the TLR9 signaling pathway. [ABSTRACT FROM AUTHOR]

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]

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5ʹ-untranslated region (UTR) of 52 bp, a 3′-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses. • The molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp. • The CiPrx2 mRNA was ubiquitously expressed in the various tissues. • CiPrx2 was likely to be involved in the immune response against viral and bacterial infection. • Subcellular localization of CiPrx2-GFP fusion protein were only distributed in the cytoplasm. • CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity and protect DNA against oxidative damage. [ABSTRACT FROM AUTHOR]

Autophagy is an essential and conserved process that plays an important role in physiological homeostasis, adaptive response to stress and the immune response. Autophagy-related proteins (ATGs) are key components of the autophagic machinery. In the study, grass carp (Ctenopharyngodon idella) autophagy-related gene 5 (ATG5) and 12 (ATG12) were identified. In the gill and intestine, ATG5 and ATG12 were highly expressed, but after grass carp reovirus (GCRV) infection, they were decreased significantly. In Ctenopharyngodon idella kidney (CIK) cells, the sharp variation of ATG5 and ATG12 expression was observed after poly(I:C) infection. Subcellular localisation showed that ATG5 and ATG12 were evenly distributed in the cytoplasm and nucleus. However, the interaction between ATG5 and ATG12 was only found in cytoplasm in both 293T cells and CIK cells. In addition, the overexpression of ATG5 or ATG12 in 293T cells showed enhanced autophagy, and autophagic process was facilitated when ATG5 and ATG12 were simultaneously overexpressed. Dual-luciferase activity assay indicated that both ATG5 and ATG12 remarkably suppressed the promoter activity of IRF3 , IRF7 , and IFN-I. Further, ATG5 and ATG12 conjugate showed far stronger inhibitory affection on the expression of IFN-I than either ATG5 or ATG12 in response to poly(I:C) or GCRV infection. Taken together, the results demonstrate that grass carp ATG5 and ATG12 play an important role in innate immunity and autophagy. • The grass carp ATG5 and ATG12 were identified and analysed. • ATG5 interacted with ATG12 in CIK and 293T cells. • ATG5 and ATG12 proteins promoted autophagy. • ATG5 and ATG12 repressed the promoter activity of IRF3 , IRF7 and IFN-I. • ATG5 and ATG12 conjugate suppressed the expression of IFN-I signaling genes. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) family are known as an important antioxidant enzyme as the first line of defense against oxidative damage, and also involved in immune responses following viral and bacterial infection. Here, a full-length Prx1 cDNA sequence (CiPrx1) was cloned from grass carp (Ctenopharyngodon idella), which was 1029 bp, including a 5′-terminal untranslated region (UTR) of 121 bp, a 3′-UTR of 272 bp, an open reading frame of 600 bp encoding 199 amino acids with molecular weight of 22.21 kDa and isoelectric point of 6.30. CiPrx1 shares 80.8–99% protein sequence similarity with Prx1 of other fishes. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of CiPrx1; this indicated that it was a member of 2-Cys Prx. Subcellular localization of CiPrx1 was only strongly distributed in the cytoplasm. Quantitative real-time PCR (RT-qPCR) assays revealed that CiPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in liver, gill and spleen. Further, the expression of CiPrx1 can be induced by grass carp reovirus (GCRV), lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly I:C) infection in the different tissues. Moreover, the recombinant CiPrx1 (rCiPrx1) protein was found a potential antioxidant enzyme, that could inhibit DNA damage from oxidants. Altogether, our results imply that CiPrx1 is associated with defending against virus and bacteria pathogens and oxidants in grass carp. • Peroxiredoxin 1 from grass carp was cloned and characterized. • CiPrx1 mRNAs was ubiquitously detected in all tested tissues. • The expression of CiPrx1 can be induced by GCRV, LPS and Poly I:C infection in the different tissues. • Subcellular localization of Prx1-pEGFP was only strongly distributed in the cytoplasm. • The antioxidant activity of the recombinant rCiPrx1 was assessed by mixed-function oxidase assay. [ABSTRACT FROM AUTHOR]

Highlights: [•] Grass carp TRIF exhibits sequence divergence from its orthologs. [•] CiTRIF might activate IFN in distinct manner from that in mammals. [•] CiTRIF plays a critical role in RLR pathway in antiviral immune response. [•] CiTRIF inhibits GCRV replication in CIK cells. [ABSTRACT FROM AUTHOR]