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Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 (ptpn6) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp (Carassius gibelio) ptpn6 homeologs (Cgptpn6-A and Cgptpn6-B), each of which had three alleles with high identities. Then, relative to Cgptpn6-B , dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that Cg SHP1-A (encoded by the Cgptpn6-A gene) and Cg SHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of Ca TBK1-induced phosphorylation of Ca MITA shared by Cg SHP1-A and Cg SHP1-B, and the autophagic degradation of Ca MITA exclusively by Cg SHP1-A. Meanwhile, the data support that Cg SHP1-A and Cg SHP1-B have sub-functionalized and that Cg SHP1-A overwhelmingly dominates Cg SHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates. [ABSTRACT FROM AUTHOR]

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs (Cgviperin-A and Cgviperin-B) in auto-allo-hexaploid gibel carp (Carassius gibelio). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both Cg Viperin-A and Cg Viperin-B were found to interact with crucian carp (C. auratus) herpesvirus (Ca HV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, Cg Viperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in Cg Viperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of Cg Viperin-A and Cg Viperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of Cg Viperin-A and Cg Viperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes. [ABSTRACT FROM AUTHOR]

A strain of Clostridium butyricum (Cb) was isolated and characterized as an immunostimulant for gibel carp. Healthy gibel carp were fed and immersed with or without Cb (group Cb + V and group V), and then were intraperitoneally injected with Carassius auratus herpesvirus (CaHV) respectively. The fish began to die at 4 days post injection (dpi). The overall survival rate of group Cb + V (45%) is more than that of group V (11%), which indicated the significant protection effect of Cb. The RT-qPCR results showed that virus major capsid protein gene (MCP) copy numbers of group Cb + V was significantly lower (P < 0.05) than that of group V at 3 dpi. Meanwhile, the expression levels of the four host innate immunity-related genes (IL11, IRF7, PKR, and Mx) were significantly higher (P < 0.01) in group Cb + V than in group V at this time point. IRF7 and PKR also had significantly higher expression levels in group Cb + V than in group V at 0 dpi, which revealed enhanced immune responses in Cb-treated fish. Treatment with Cb prompted stronger host immune responses at the early stage of virus infection, and then resulted in the survival of gibel carp. To our knowledge, it is the first report about the immune protection of fish from herpesvirus by probiotics. [ABSTRACT FROM AUTHOR]

Background: Accompanied with rapid growth and high density aquaculture, gibel carp has been seriously threatened by Carassius auratus herpesvirus (CaHV) since 2012. In previous study, distinct CaHV resistances and immune responses were revealed in the diseased individuals of three gibel carp gynogenetic clones (A+, F and H). However, little is known about the gene expression changes in the survivors after CaHV challenge, particularly their differences of innate and adaptive immune system between susceptible clone and resistant clone. Results: We firstly confirmed the CaHV carrier state in the survivors of three gibel carp clones after CaHV challenge by evaluating the abundances of five CaHV genes. The assay of viral loads indicated the resistant clone H possessed not only stronger resistance but also higher tolerance to CaHV. Then, 2818, 4047 and 3323 differentially expressed unigenes (DEUs) were screened from the head-kidney transcriptome profiles of survivors compared with controls from clone A+, F and H. GO and KEGG analysis suggested that a persistent immune response might sustain in resistant clone H and F, while susceptible clone A+ had a long-term impact on the circulatory system which was consistent with the major symptoms of bleeding caused by CaHV. Among the top 30 enriched pathways of specifically up-regulated DEUs in respective clones, 26, 7 and 15 pathways in clone H, F and A+ were associated with infections, diseases, or immune-related pathways respectively. In addition, 20 pathways in clone F belonged to "metabolism" or "biogenesis", and 7 pathways involved in "circulatory system" were enriched in clone A+. Significantly, we revealed the differential expression changes of IFN system genes and immunoglobulin (Ig) genes among the survivors of three clones. Finally, myosins and Igs were identified as co-expression modules which were positively or negatively correlated to CaHV viral loads respectively. Conclusions: Our results revealed the common and distinct gene expression changes in immune and circulatory system in the survivors of three gibel carp gynogenetic clones with different CaHV resistances. The current study represents a paradigm of differential innate and adaptive immune reactions in teleost, and will be beneficial to the disease-resistance breeding of gibel carp. [ABSTRACT FROM AUTHOR]

• Gibel carp IFNa and IFNc display significantly antiviral potential to viral infection. • ISRE motifs within promoters are responsible for the induction of carp IFN and IFNc by diverse stimuli including themselves. • Gibel carp IFNa and IFNc have different-sized 3′UTR that is a potential factor for regulation of IFN response. Fish interferon (IFN)-mediated antiviral innate immunity is the first line of defense against virus invasion. In the present study, we identify two fish IFN genes (here tentatively named IFNa and IFNc) with different-sized 3′ UTRs from clone F strain of gibel carp Carassius auratus gibelio. Carp IFNa has a relatively short 3′UTR without AU-rich elements (AREs) but IFNc has a long one with 9 AREs. Functionally, carp IFNa and IFNc display significantly antiviral potential to viral infection, likely through induction of downstream IFN-stimulated genes (ISGs). Both carp IFN genes are induced by viral infection, poly(I:C) treatment and IRF3/7, which are ascribed to the IFN-sensitive response elements (ISRE) within their promoters. Carp IFN genes are also induced by each other and by themselves, indicating existence of a positive feedback loop in fish IFN-mediated antiviral immune response. Comparative analyses of 3′UTR-mediated expression regulation at mRNA and protein levels show that the ARE-containing 3′UTR of carp IFNc rather than the short 3′UTR of carp IFNa promotes mRNA decay but instead results in high-level protein expression, indicating that 3′UTR of fish IFN mRNAs might be a potential factor for regulation of IFN-mediated antiviral immune response. Considering a fact that a given protein function is largely related to its protein level, these results suggest that both promoter and 3′UTR contribute to the transcription and translation of fish IFN genes, thus shaping their eventually antiviral potential. [ABSTRACT FROM AUTHOR]

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus ( Ca HV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A + , F and H were related to the susceptibility to Ca HV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon ( IFN ) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs . [ABSTRACT FROM AUTHOR]

Viperin is increasingly thought as a key protein in antiviral response. However, the in vivo antiviral effects of fish viperin remains unresolved. Hexaploid gibel carp (Carassius gibelio) is an important aquaculture species in China and its culture industry has suffered enormous economic losses due to an epizootic disease caused by C. auratus herpesvirus (Ca HV) since 2012. In our previous studies, two duplicated viperin homeologs (Cgviperin-A and Cgviperin-B) were identified and their divergent antiviral mechanisms were revealed through in vitro overexpression in CAB (C. auratus blastulae embryonic) cell. Here, we tried to explore their function difference in vivo by using CRISPR/Cas9 editing. Firstly, we uncovered their biased expression patterns and differential promoter activities. Then we mutated them singly or simultaneously in resistant clone H of gibel carp by using CRISPR/Cas9 and finally confirmed that they cooperatively participated in the antiviral battle between gibel carp and Ca HV through virus challenge and comparative transcriptome analysis. Cg Viperin-A depletion mainly affected immune-related pathways and Cgviperin-B deficiency influenced autophagy-associated pathways. Simultaneous disruption of them resulted in complete loss of resistance ability of gibel carp against Ca HV. Significantly, the deficiency of Cgviperin-A influenced the expression of Cgviperin-B in Cgviperin-A −/−/− mutant, but Cgviperin-A transcripts kept a high level of expression in the Cgviperin-B −/−/− mutant, same as in the WT. Therefore, the current findings are not only the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo, but also explain the detailed evolutionary divergence of a duplicated gene in a recurrent polyploid fish. • Two duplicated viperin homeologs were edited singly or simultaneously in hexaploid gibel carp by using CRISPR/Cas9. • It is the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo. • Cg Viperin-A and Cg Viperin-B have sub-functionalized, Cg viperin-B has evolved a novel function related to autophagy. • Cg Viperin-A and Cg Viperin-B cooperatively participate in the antiviral response of gibel carp against CaHV. [ABSTRACT FROM AUTHOR]