Screening And Functional Analysis Of Crucial Cellular MiRNA In Chicken Macrophages Infected With Newcastle Disease Virus Virulent Strain

Newcastle disease(ND)caused by Newcastle disease virus(NDV)is an acute and highly contagious disease.Because of its mortality rate to susceptible poultry is up to 100%,ND is listed as a must be reported animal epidemic disease by the World Animal Health Organization(OIE),belongs to the first category of animal diseases of the Ministry of Agriculture and Rural Affairs and General Administration of Customs.However,NDV avirulent strain does not cause lethal infection in poultry,but only causes relatively mild clinical manifestations of respiratory system or intestinal infection without clinical symptoms,so it is often used for vaccination.Although the different degrees of host immune response,body pathological damage,and cytokine storm caused by virulent and avirulent NDV strains infecting the body or cells can be observed clinically,the specific mechanism of the difference in the process of NDV virulent and avirulent strains infecting the host has not been fully revealed.microRNA(miRNA)is a kind of endogenous single stranded non-coding smallRNA with a length of 18 ～ 25 nt,which widely exists in animals and plants.It can degrades or interferes with the transcription of target gene mRNA by base complementation,and then regulates the expression of target gene.In the game between virus and host,it has been proved that a variety of miRNAs can perform important functions in the infection and replication differences of different virulence viruses through different mechanisms.However,it is still blank whether miRNAs play an important role in the process of host infection with virulent and avirulent NDV.Therefore,screening and functional analysis of crucial differential host miRNAs in the process of virulent and avirulent NDV infection of host cells will help to clarify the differential mechanism in the process of virulent and avirulent NDV infection of host cells,lay a theoretical foundation for revealing the pathogenic mechanism of virulent NDV infection,and provide new ideas for the prevention and control of ND from the perspective of miRNA.The main contents of this thesis are divided into the following three parts:1 Analysis of differential miRNA expression profiles in chicken macrophages infected with Newcastle disease virus virulent and avirulent strainIn this study,chicken macrophages were infected with NDV virulent and avirulent representative strains(virulent strain NA-1 of genotype VII and avirulent vaccine strain La Sota of genotype II)with MOI=0.1,2 and 5 respectively,the cell supernatant and cellRNA of infected cells were collected at 6,12,24,48 and 72 hours after infection,then use q PCR,virus titer determination and immunofluorescence experiments to measure virus proliferation and cytopathic conditions to evaluate the establishment of cell model of virus infection in vitro.The results showed that the infection dose of MOI=2 can effectively proliferate NDV in cells with virulent and avirulent strains.However,when the infection dose is 0.1 and 5,the time of cell lesions is later or earlier,and the degree of cytopathic changes is lighter or more severe.Therefore,samples of chicken macrophages infected with MOI = 2 for 24 and48 hours were used for high-throughput sequencing in this study.High-throughput data showed that compared with the avirulent La Sota infection group,there were 130(up-regulated 87,down-regulated 43)and 196(up-regulated 68,down-regulated 128)differentially expressed miRNAs after 24 h and 48 h infected with virulent NA-1,respectively,and 33 miRNAs were persistently differentially expressed;10 miRNAs were randomly selected for q PCR verification,and the results were basically consistent with the results of high-throughput sequencing.2 Screening of crucial miRNA and identification of its target genes in chicken macrophages infected with Newcastle disease virus virulent strainIn order to explore the function of differentially expressed miRNAs in the process of virulent and avirulent NDV infection,based on whether the target genes of miRNAs are involved in virus infection or immune process,biological software was used to screen the crucial miRNAs in the process of virulent and avirulent NDV infection and verify their target genes.The results showed that a total of 12 miRNAs related to virus infection or immune process were screened out of 33 persistently differentially expressed miRNAs obtained from the virulent and avirulent NDV infection obtained in the previous stage.Further study showed that compared with the uninfected group,only the expression levels of gga-novel-217-5p and its target gene TAK1 binding protein 1(TAB1)were significantly changed after NDV virulent strain infection,but not in the NDV avirulent strain infection group.Meanwhile,compared with gga-novel-217-5p unrelated sequence,the expression of TAB1 was significantly up-regulated by gga-novel-217-5p inhibitor transfection,and downregulated by mimic transfection.However,the expression of TAB1 was not changed by gga-novel-217-5p mutant transfection,which confirmed that gga-novel-217-5p does have a targeting relationship with TAB1.In conclusion,this chapter successfully screened the crucial miRNA gga-novel-217-5p and its target gene TAB1 in the process of virulent and avirulent NDV infection,indicating that the gga-novel-217-5p/TAB1 axis plays an important role in the process of NDV virulent strain infected chicken macrophages.3 Functional analysis of crucial miRNA in chicken macrophages infected with Newcastle disease virus virulent strainStudies have shown that AK1-binding protein 1(TAB1)are known as two crucial upstream mediators in the NF-κB signaling pathway.TAK1 interacts with TAB1 to form the TAK1–TAB1 complex,which triggers NF-κB activation through a series of cascade reactions,thereby regulating the expression of downstream cytokines.This chapter aims to explore the function and mode of action of the gganovel-217-5p/TAB1 axis in the process of NDV virulent strain infection.Start with the levels of differentially expressed cytokines in chicken macrophages infected with NDV virulent and avirulent strain.Transfection of gga-novel-217-5p inhibitor can significantly up-regulate the expression levels of cytokines IL-1β,iNOS,TNF-α and IFN-α in chicken macrophages.Further experimental results confirmed that the gganovel-217-5p/TAB1 axis activates the NF-κB signaling pathway by regulating the phosphorylation level of NF-κB inhibitor IκB-α.In conclusion,this chapter confirmed that down-regulating the expression of host gga-novel-217-5p by NDV virulent strain infected chicken macrophages,which led to the up-regulation of TAB1 expression level,and then promoted IκB-α phosphorylation to activate NF-κB signaling pathway,leading to a higher level of cytokine expression.