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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]

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]

Abstract Grass carp, an economically important aquaculture fish, is very sensitive to Grass Carp Reovirus (GCRV). Haemorrhagic disease caused by GCRV infection can cause large-scale death of first-year grass carp, thereby severely restricting the intensive culture. Serpins (serine protease inhibitors) belong to the protease inhibitor gene family and are involved in numerous physiological and pathological processes, particularly coagulation and anticoagulation. Reports on grass carp serpins are scarce. Thus, we cloned six grass carp serpin genes (serpinb1 , serpinc1 , serpind1 , serpinf1 , serpinf2b and serping1) in this study. Molecular evolution showed that serpins between grass carp and zebrafish or carp are the closest relatives. SERPIN domains in these 6 serpins and reactive centre loop (RCL) along with their cleavage sites of 5 serpins (serpinb1 , serpinc1 , serpind1 , serpinf2b and serping1) were predicted. Real-time quantitative PCR (RT-qPCR) showed that these serpins displayed tissue significance. Among them, serpinc1 , serpind1 , s erpinf2b and serping1 had the highest expression levels in the liver. After GCRV infection, RT-qPCR showed that the liver-enriched serpins were significantly changed. Key procoagulant factor genes (kng-1 , f2 , f3a , f3b and f7) and anticoagulant genes (tpa , plg , thbd , proc and pros) also showed significant changes on the mRNA level. Comprehensive comparative analysis showed that the up-regulated expression of key clotting factor genes was more prominent than that of main anti-coagulation factor genes. Thus, the function of coagulation may be more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. The serpins were involved in GCRV infection and liver-enriched serpins participate in the interaction between coagulation and anticoagulation. This study provided new insights into further research on the biological functions of grass carp serpins and clarifying the molecular mechanism of GCRV affecting the homeostasis of grass carp blood environment. Highlights • Six serpin genes in grass carp (Ctenopharyngodon idella) were cloned, and all the serpin proteins contained an SERPIN domain. • Liver-enriched serpins (serpinc1 , serpind1 , serpinf2b and serping1) may participate in the interaction between coagulation and anticoagulation. • The function of coagulation was more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. • Serpins may affect the homeostasis of blood environment in grass carp during GCRV infection. [ABSTRACT FROM AUTHOR]

Grass carp ( Ctenopharyngodon idellus ) is an economical aquaculture species in China, and the Grass Carp Reovirus (GCRV) that causes hemorrhagic disease seriously affects the grass carp cultivation industry. Substantial evidence indicates that there is an association between the membrane-associated RING-CH family of E3 ligase (MARCH) family and immune defense in mammals, while functional studies on non-mammalian MARCH proteins are limited. In order to know the characteristics of the MARCH genes in C. idellus , eight MARCH genes ( MARCH1, 2, 5, 6, 7, 8, 9 and 11 ) were cloned and the open reading frames (ORF) were identified in grass carp. All MARCH proteins in grass carp contained an RING-CH domain, which is characteristic of the MARCH protein. The phylogenetic analysis revealed that different MARCH proteins gathered into their separate clusters. All eight members of the MARCH gene family were detected in all tissues sampled, but the relative expression level differed. In addition, the mRNA expression of all the MARCHs was regulated at different levels in the immune organs after a GCRV challenge, and they responded robustly in both the intestine and liver. The mRNA expression of MARCH8, MHC II, TfR, IL1RAP, EGR1, and DUSP1 in the intestine after GCRV infection was analyzed, and the results showed that MARCH8 could negatively regulate TfR, IL1RAP, EGR1, and DUSP1, which signaled via the MAPK or NF-κB-activation pathways that play vital roles in immunity. Our findings identified a novel gene family in C. idellus and provided novel evidence that MARCH genes are inducible and involved in the immune response. Moreover, MARCH8 might function to negatively regulate immune receptors in C. idellus . Therefore, the MARCH might play a vital role in regulating the immune response of C. idellus . [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]