Preliminary Study On The Effect Of Interferon Regulatory Factors On Tilapia Lake Virus Propagation In Tilapia

Tilapia(Oreochromis niloticus)is one of the most important freshwater farmed fish in China and whole the world with highly economic value.However,diseases are frequently emerging along with the continuous expansion of culture scale.In recent years,Tilapia Lake virus disease(TiLVD)caused by Tilapia Lake virus(TiLV),has posed a serious threat to the tilapia industry in several countries around the world.Effective control measures are still lacking due to poor understanding on the pathogenesis of TiLV and the immune defense mechanism of the host.The innate immune response induced by pattern recognition receptors to recognize pathogenassociated molecular patterns is the first line of defense of the organism against invasion by foreign pathogenic microorganisms.Among them,the type I interferon(Type Ⅰ interferon,IFN-Ⅰ)system of fish plays a key role in the innate immune process.Interferon regulatory factor(IRF),a key class of transcription factors involved in regulating interferon production,also plays an important role in antiviral immunity.However,the antiviral effects of IRFs from tilapia have not been reported yet.This study aimed to provide an important reference for the antiviral mechanism of tilapia by identifying the function of tilapia IRFs during TiLV infection.These results of this study are as follows.1.Gene cloning and bioinformatics analysis of tilapia IRFsIn this study,six members of tilapia IRFs,including IRF1,IRF2,IRF4,IRF7,IRF8 and IRF9,were amplified by gene cloning.Structural domain analysis showed that the structures were highly similar to other species,which all contained a conserved IRF structural domain.Multiple sequence comparisons showed that IRFs have conserved DNA structural domains,serine-rich C-termini and tryptophan disability clusters.Phylogenetic tree analysis indicated that the IRF family can be divided into four subfamilies: IRF1 subfamily(IRF1 and IRF2),IRF3 subfamily(IRF7),IRF4 subfamily(IRF4,IRF8 and IRF9)and IRF5 subfamily.2.Tissue distribution and subcellular localization analysis of tilapia IRFs genesTissue distribution analysis revealed that all six IRFs genes were widely distributed in tilapia tissues using Real-time PCR.The expression of IRF1 was highest in gill and intestine,but relatively low in brain and liver.The expression of IRF2 was high in intestine and head and kidney,but relatively low in skin and heart.The expression of IRF4 was high in spleen and gill,but relatively low in liver,middle kidney and heart.The expression of IRF7 was high in gill and intestine,but relatively low in liver,middle kidney and heart.The expression of IRF7 was higher in tissues such as gill and intestine,while the expression in head and kidney was relatively low.IRF8 was higher in tissues such as gill and spleen,while the expression in tissues such as middle kidney and muscle were relatively low.IRF9 was higher in tissues such as head and kidney and heart,while the expression in tissues such as skin and stomach were relatively low.The p EGFP-N1 recombinant vector of IRFs was constructed.Transfection into tilapia brain cells(TiB)cells followed by observing under laser confocal microscope showed that IRF1,IRF4 and IRF7 were mainly distributed in the cytoplasm of TiB cells,IRF8 and IRF9 were mainly distributed in the nucleus,while IRF2 was distributed both in the cytoplasm and nucleus of TiB cells.3.Effects of overexpression of IRFs from tilapia on TiLV proliferation.After transfection of pEGFP-IRFs recombinant plasmid into TiB cells infected with TiLV,relative Real-time PCR assay revealed that the relative expression of viral S8 fragment in the six IRFs overexpression group was significantly lower than that in the control group at 24 h and 48 h of infection.Absolute Real-time PCR further confirmed that the viral genome copy number of the six IRFs overexpression was also significantly lower than that of the control group after 24 h and 48 h of infection.The results indicated that all six tilapia IRFs significantly inhibited the proliferation of TiLV.4.Modulation of IFN promoter activity by the genes of tilapia IRFs:The recombinant plasmids of IRFs with flag tags were constructed and cotransfected with the tilapia IFNa3 promoter to TiB,respectively,to detect changes in the upstream promoter IFNa3 activity.The results of dual luciferase reporter showed that all six IRFs genes significantly activated IFNa3 promoter activity after overexpression.In contrast,IFNa3 promoter activity was significantly lower after TiLV infection than in uninfected cells,indicating that TiLV may inhibit the interferon effect activated by IRFs.In summary,this study tentatively showed that tilapia IRFs can trigger the innate immune response pathway involved in downstream type I interferon by promoting upstream interferon promoter activity,which in turn inhibits TiLV proliferation,while TiLV may promote its own replication by inhibiting the activating interferon effect exerted by IRFs,providing a theoretical basis for antiviral immunity studies in tilapia.