Cloning And Expression Of Duck DDX3X Gene And Its Anti-duck Tembusu Virus Activity

Duck Tembusu virus(DTMUV)is a newly emerged pathogenic flavivirus mainly harmful to ducks.It has the characteristics of fast transmission,strong infection,high incidence rate.The virus can infect not only ducks of various breeds,but also chickens,geese and wild birds,causing great economic losses to China's duck-breeding industry.The innate immune system is the host's first line of defense against infection by pathogenic microorganisms.DDX3 X protein as an important member of the dead box RNA helicase family.DDX3 X can regulate RNA metabolism and researches on its participation in host innateimmunity have been increasing in recent years.Reports show that DDX3 X can act as a recognition receptor for viral nucleic acids,interact with adaptor proteins of innate immune signal pathways,promote the production of type I interferon(IFN),and also interact with important transcription factors in immune signal pathways to regulate the expression of I-IFN and affect viral replication.In recent years,with the increasing scale and quantity of duck farming,the duck farming industry has developed rapidly in our country,but facing the huge pressure of disease prevention and control.Especially in recent years,new infectious diseases of ducks have emerged continuously,seriously endangering the healthy development of duck farming.The severity of the epidemic disease is closely related to pathogen's pathogenicity of the and the interaction between the pathogen and the host immune response.Compared with mammals,there is less research on the innate immune system of waterfowl.The identification of innate related genes of waterfowl including ducks and geese and the interaction between the innate related genes and viruses need to be further strengthened.So far,the information about DDX3 X protein in ducks is not available.Therefore,this study first cloned and identified duck DDX3X(duDDX3X)gene from spleen samples of Cherry Valley duck and conducted a preliminary study on the interaction between it and DTMUV.The results showed that the duDDX3 X genome consists of 1959 bases and encodes 652 amino acids.duDDX3 X has the typical structure of the family,including 9 motifs,Q?I?Ia?Ib?II?III?v and VI,DEAD domain and HELICc domain.Amino acid homology analysis showed that the amino acid sequence of duDDX3 X had the highest homology with chicken,98.3%.The homology with goose,human,mouse and other mammals is about 93%,while the homology with fish is the lowest(80.2%).Real-time quantitative real-time PCR(qPCR)analysis shows that duDDX3 X has a broad-spectrum in tissue expression,and is commonly expressed in the heart,liver,spleen,lung,kidney,brain,cerebellum,brainstem,pancreas,myogastric,glandular stomach,duodenum,jejunum,ileum,cecum,rectum,bursa of fabricius,thymus,esophagus,trachea and muscle of healthy ducks,especially in cecum,with the highest expression level,and is also strongly expressed in ileum,liver,spleen,pancreas,rectum and jejunum.However,the expression level in kidney,lung,brain,cerebellum and bursa of Fabricius is relatively low.Subcellular localization analysis showed that duDDX3 X protein was mainly distributed in cytoplasm.Through qPCR and western blot analysis,we found that the virus can significantly downregulate the expression of duDDX3 X in duck embryo fibroblasts(DEF)infected by DTMUV,suggesting that duDDX3 X may participate in virus replication.DTMUV was inoculated in DEF cells overexpressing duDDX3 X,and then cell samples were collected at different time points to detect virus titer.We found that duDDX3 X could significantly inhibit the replication of DTMUV in the early stage of infection(24 h and 36 h).Furthermore,the replication of DTMUV increased after the interference of duDDX3 X,indicating that the duDDX3 X protein can effectively inhibit the replication of DTMUV.In order to further determine the mechanism of duDDX3 X inhibiting virus replication,we tested the effect of duDDX3 X on IFN-?,NF-?B,and IRF-7 promoter activity through double luciferase reporter gene experiment,and found that duDDX3 X can significantly promote IFN-? and IRF-7 promoter activity,and the detection of host cytokines after duDDX3 X overexpression also confirmed that duDDX3 X protein can significantly promote the expression of I-IFN,but has no significant effect on the expression of proinflammatory cytokines such as IL-1,IL-6,CXCL-8,etc.duDDX3 X was overexpressed or knockdown,cells were infected with DTMUV atfer 12 h,and detected IFN-? content 24 h after infection.It was found that duDDX3 X overexpression can significantly promote DTMUV stimulated IFN-? expression,while the amount of DTMUV stimulated IFN-? expression decreased after duDDX3 X interference,suggesting that duDDX3 X may inhibit virus replication by promoting the expression of I-IFN after DTMUV infection.duDDX3 X and RIG-I were co-transfected into cells and their effects on IFN-promoter activity were detected.The results showed that duDDX3 X could also promote IFN-? expression induced by RIG-I,indicating that duDDX3 X protein could regulate I-IFN expression through RLR signaling pathway and affect viral replication.In summary,the duDDX3 X gene was first cloned in this study.The gene has the highest homology with chicken DDX3 X and has a extensive tissue expression profile.Studies on the interaction between DTMUV and duDDX3 X show that DTMUV can downregulate the expression of duDDX3 X,but duDDX3 X can induce the expression of I-IFN and inhibit viral replication by promoting the production of I-IFN stimulated by DTMUV and RIG-I.The research results are helpful to understand the role of DDX3 X in duck's innate immune system,and lay a foundation for further research on the innate immune response between DTMUV and host.