Isolation And Identification Of Chicken Exosomes And Effect Of Virus Components On Replication Of NDV

Newcastle Disease(ND)is an important infectious disease of poultry,waterfowl and wild birds caused by Newcastle Disease virus(NDV).It is also one of the five major types of animal diseases that are mainly controlled in the 13 th Five-Year Development Plan of China's animal husbandry and the National Medium-and Long-Term Animal Disease Control Plan(2012-2020).At present,it is known that NDV HN and F protein determine virus virulence,NP,P,L and M protein also play a role in the process of virus infection.In addition,NDV infection also trigger apoptosis and autophagy,affecting the transmission of the virus between cells.However,the pathogenic of NDV has not been fully explained,with the expanding of biological research field,the mechanisms of NDV infection,proliferation and pathogenesis are constantly supplemented and improved.Exosomes are vesicles secreted by cells with a diameter of 30-120 nm.Exosomes can transfer lipids,proteins and genetic materials to the recipient cells,thus forming an intercellular information transmission system with exosomes as a bridge and participating in cell migration,cell proliferation,cell activation or tissue damage repair.Studies have shown that exosomes are closely related to virus infection and host antiviral immune response,and exosomes can play a role in the process of virus infection by carrying complete infectious virus particles or viral components.At present,the relationship between NDV infection and exosomes is still unclear.Therefore,the exosomes was taken as the starting point in this study,through the detection of viral components in exosomes and analysis of the influence of exosomesin the process of virus infection,the interaction between exosomes and NDV is clarified.The premise of studying the biological function of exosomes is to obtain high-purity exosomes,which are similar to NDV particles in physical properties,so they cannot be separated by conventional methods.Therefore,in this study,we firstly established a method of separation of avian exosomes using proteinA/G magnetic beads combined with avian CD63 antibody and detected the products.Western Blot results showed that the product expressed the exosome marker protein TSG101,transmission electron microscopy observation showed that the product had a typical disc-shaped exosome form with a diameter of about 40 nm,indicating that the avian source NDV-related exosome(NDV Ex)was successfully obtained.Furthermore,This study further examined the infectivity of NDV Ex and the viral components in NDV Ex.Q-pcr and Westernblot test results showed that NDV Ex contained W,V and F proteins,but there was no HN,M proteins and viral genome.To further explore the effect of NDV Ex on NDV proliferation in macrophages.The results showed that the effect of NDV Ex on virus replication was related to the duration of infection and the initial titer of the virus.During MOI=0.1,NDV ex gene had no significant change at 3-6h,NP gene level slightly decreased at 9h,significantly decreased at 12 h,and significantly increased at24h;during MOI=1,NDV Ex had no effect on NDV at 3h,down-regulated the level of NP gene at 6-9h,almost recovered at 12 h,and showed up-regulation at 24 h.The results showed that NDV Ex began to inhibit virus replication at 6h,recovered steadily at 12 h,and promoted virus proliferation at 24 h.Through the detection of activated molecules and antiviral cytokines of chicken macrophages(HD11),it was found that NDV Ex induced activation of HD11 in the early stage,simultaneously promoted the expression of IFN-? and ISGs,and inhibited the replication of NDV,and later inhibited the activation of HD11 and the expression of IFN-? and ISGs to promote the replication of NDV.The above results suggest that immune activation is caused by the activationof macrophages in the early stage of NDV Ex,which provides convenience for the replication and transmission of NDV in the later stage.To sum up,this paper first isolated NDV-related exosomes and analyzes the viral components present in them,further detected the effect of NDV Ex and viral proteins on the proliferation of NDV,preliminarily explored its mechanism in the process of virus infection.The results showed that,NDV Ex regulated the activation of macrophages and the expression of antiviral cytokines in the process of virus infection,indicating that exosomes are important pathways involved in NDV infection and replication.