| Newcastle disease(ND)caused by a virulent strain of Newcastle disease virus(NDV)is an acute highly contagious infectious disease of poultry,which is characterized by elevated body temperature,dysentery,injury in nerve system,respiratory disorders,gastrointestinal and respiratory haemorrhage as clinical symptoms.Over 240 bird species including poultry and some mammals can be infected by NDV.The disease is widespread all over the world and has brought huge economic losses to poultry industry.Active immunization measures have been taken to prevent and control ND in China,which has controlled the spread of ND in a large scale.However,due to the poor biosafety system in our country and failure of NDV immunization in farms,local outbreaks of ND occur from time to time.Therefore,it is urgent deeded to explore new strategies to prevent and control ND.Chicken Melanoma Differentiation Association antigen 5(chMDA5)is one of pattern recognition receptor(PRR)that sensor exogenous virus infection in chicken cells,monitoring NDV infection,mediating the expression of interferon,triggering innate immunity to against virus infection in chickens.To explore the regulation of chMDA5 on NDV infection and immunity is helpful to elucidate the mechanism of anti-virus and vaccine efficacy from the perspective of chicken body,providing new information for revealing the interaction between virus and body,and providing new references for improving the efficacy of NDV vaccine.The main researches and the results are as follows:1.Study on chMDA5 inducing the expression of Interferon in VitroThe expression of chMDA5 and IFN-?in DF-1 cells infected with NDV virulent strain F48E9 and attenuated strain La Sota by 0.1 MOI was detected by RT-q PCR at the time of 8h,12 h,24 h and 48 h post infection.It was found that the expression of chMDA5 in DF-1cells was up-regulated by infection of both F48E9 or La Sota.The expression of IFN-?was up-regulated in DF-1 cells infected with F48E9,while up-regulation in DF-1 cells infected with La Sota was only at 24 hours post infection.The expressions of chMDA5 and IFN-?in the lungs of SPF chickens infected with F48E9 and La Sota by 10~5 PFU per bird were detected by RT-q PCR.It was found that the expressions of chMDA5 and FN-?in chicken lungs were up-regulated by infection of F48E9 or La Sota.The expression of chMDA5 in spleens of chickens infected with F48E9 was up-regulated post infection,and the expression of IFN-?was down-regulated and then up-regulated,reaching normal level finally.The expression of chMDA5 in the spleens of chickens infected with La Sota was down-regulated at 12 hours post infection,and up-regulated at 24 and 48 hours post infection.The expression of IFN-?in the spleens of chickens infected with La Sota was down-regulated.Over-expression of chMDA5 on DF-1 by eukaryotic expression plasmid with chMDA5 showed that the expression of IFN-?was significantly induced and the proliferation of NDV strain F48E9 was significantly inhibited by over-expression of chDMA5 in vitro.Inhibiting the expression of chMDA5 in DF-1 cells by RNA interference showed that the expression of IFN-?was significantly inhibited by inhibiting the expression of chMDA5 in DF-1 cells,but the proliferation of NDV strain F48E9 was not affected significantly.2.Study on the functional domain of chMDA5 inducing the expression of interferonChMDA5 consists of 1001 amino acid residues.In order to study on the functional domain of chMDA5 inducing the expression of interferon,it was divided into 5 segments of different lengths:1-328aa,1-483aa,1-642aa,325-1001aa and full length according to the structure of chMDA5.Five eukaryotic expression vectors expressing different fragments of chMDA5 were constructed using pCMV-3HA as bone structure.Transfected into DF-1 cells,all of the five fragments could be expressed in DF-1 cells detected by western blot.The expression of IFN-?in chickens was detected by RT-q PCR.It was found that the expression of IFN-?was induced by the fragments containing caspase activation and recruitment domain(CARD)domain.There was no significant difference in the expression level of IFN-?induced by 1-483 aa and 1-642 aa fragments,and there was no significant difference in the expression level of IFN-?induced by 1-483 aa,1-642 aa or chMDA5.So,1-483 aa of chMDA5 could be used as functional domain to study the expression of IFN-?induced by chMDA5.3.The study on chMDA5 affecting the pathogenicity of NDV to chickenRecombinant adenovirus rAd-MDA5 expressing 1-483aa of chMDA5 and rAd-shM5interfering the expression of chMDA5 were constructed and obtained.The expression of chMDA5(483aa)in DF-1 cells could be derived by infection of DF-1 cells with rAd-MDA5,and the expression of chicken IFN-?was also be induced.The expression of chMDA5 and IFN-?in DF-1 cells was significantly reduced by infection of DF-1 cells with rAd-shM5,and the expression of chMDA5 and IFN-?induced by F48E9 infection was also down-regulated by rAd-shM5.The expression of chMDA5 expression in chickens was up-regulated post injection of rAd-MDA5.Compared with infection with F48E9 alone,the expression of IFN-?in chicken lungs were significantly up-regulated by co-infection with rAd-MDA5 and F48E9,virus load in lungs was reduced and the survival time of chickens post challenge was prolonged.The expression of chMDA5 and IFN-?in chickens was inhibited by injection of rAd-M5,while the survival time of chickens post infection with F48E9 was not affected significantly.4.Study on chMDA5 assisting activation of adaptive immune responseSPF chickens were immunized with recombinant adenovirus rAd-MDA5 expressing chMDA5(483 aa)and NDV inactivated vaccine.Blood samples were collected every 7 days post immunization.Antibody levels in serum were detected by hemagglutination inhibition(HI)method.It was found that chMDA5(483 aa)expressed by adenovirus rAd-MDA5enhanced the humoral immune response induced by NDV inactivated vaccine.The chicken spleens were collected 24 hours post boost immunization.The expression of IL-4,IFN-?and MHC-?were detected by RT-q PCR.It was found that chMDA5(483 aa)increased the expression of these cell mediated immune related cytokines in chickens.Chicken peripheral blood were collected before challenge and the results of lymphocyte proliferation test showed that chMDA5(483 aa)increased the stimulation index of lymphocytes in chickens co-administrated with rAd-MDA5 and NDV inactivated vaccine.Chickens were infected with virulent NDV strain F48E9 by a dose of 10~4 PFU per chicken.Morbidity,tissue lesion,virus load in organs and virus shedding post infection were reduced by co-administrated with rAd-MDA5 and NDV inactivated vaccine.Results indicates that the immune efficacy of NDV inactivated vaccine was enhanced by chMDA(483 aa).In summary,the expression of chMDA5 and IFN-?in cells and chickens post NDV infection were studied in this research.The functional domains of chMDA5 inducing the expression of IFN were studied in details.The effects of chMDA5 on NDV proliferation and the pathogenicity of NDV to chickens were evaluated.The mechanism of chMDA5(483aa)enhancing the immune efficacy of NDV inactivated vaccine was revealed.On the one hand,it provides a theoretical basis for further study of the function of chMDA5 and a basis for further study of chMDA5 to help host resist NDV infection.On the other hand,it provided a new method for enhancing the immune efficacy of NDV vaccine and a molecular adjuvant candidate for the development of new NDV vaccine. |
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