Mechanism Of Bi-directional Regulation Of Exosome In Newcastle Disease Virus Infection

Newcastle disease(ND)is caused by infection with virulent NDV strains in poultry population,which brings huge economic losses to the poultry industry.The development of novel methods to prevent and control ND has become a major scientific and production practical problem.NDV infection lead to pathological damage to mucosal and nerve tissue,while the immune system cannot effectively control the spread of the virus,even leading to immulogic disorder and weakened elimination of the virus.The exosome is a nano-size vesicle that mediates intercellular communication,which can regulate the biological activity through the transmission of proteins,nucleic acids and lipids.Recent studies have shown that the secretion of exosome and its components are closely related to viral infection and host antiviral immunity.Viral infection changes the composition of the exosome,and exploits them to transmit viral specific molecules to inhibit antiviral response,or to creat a microenvironment benefitive to their own replication.Therefore,it is very important to study the role of exosome in the process of viral infection to provide a better understanding the pathogenic mechanism of virus.However,the role and mechanism of exosome in NDV infection remains unclear.To interpret the mechanism of exosome related viral infection,exosome was isolated and identified from NDV infected cells(NDV Ex).The m RNA transcriptsome of NDV Ex viral components were also examined.The relationship between viral infection and the release of exosome was clarified,and the signaling pathway mediated by NDV infection promoting exosome release was further explored.To analyze the mechanism of the viral infection induced exosome secretion,the effects of the components and host factors of the exosome on viral infection and replication were analyzed.In this research,the mechanism of exosme mediated NDV infection was investigated to provide theoretical basis for a better understanding the mechanism of virus infection associated with exosome secretion,and to depen the understanding of NDV infection mechanism and the strategy of ND prevention.To avoid the effects of viral particles on transcriptome and virus composition analysis,we employed PEG precipitation,supercentrifugation and CD81 tagged magnetic bead to separate exosome from NDV particles and successfully generated virus-free NDV related exosome secretion(NDV Ex).After analysis,it was found that NDV Ex does not contain NDV genome,but contains some viral proteins(NP,F,W,V).On this basis,transcriptsome of NDV Ex were analyzed,and it was found that NDV infection would reduce the m RNA content in the exosome.The majority of antiviral related genes were significantly reduced in the exosome after infection,there were still several antiviral related gene m RNA.To study the role of these differentiated genes in NDV infection,seven increased antiviral related genes after NDV infections were screened and validated in HD11,DF-1 and Hela cells.In addition,due to the lack of quantitation methods of avain exosme,two genes without significant expression change was also selected and confirmed to establish a relatively quantitative fluorescence quantitative method(q PCR)for NDV Ex.The secretion of the exosme is closely related to viral infection,but the relationship between the NDV and the release of the exosome is not clear.Therefore,taking the advantage of the quantitation method,we identified the association between NDV infection and the release of exosome.The results showed that the NDV infection of both virulent and lentogenic NDV strains could promote the release exosome,and this catalytic effect presented a dose-dependent manner.In addition,the overall amount of exosome in the supernatant tends to be stable,which does not continue to rise as time pass by,revealing the dynamic balance of exosome release and ingestion.The mechanism of NDV promoted exosme release is further investigated.There are two recognized pathways of exosme secretion,ESCRT dependent and ESCRT non-dependent pathway.By analyzing the m RNA expression of several genes related to the secretion after NDV infection.It was found that the ALIX is the only gene increased in ESCRT pathway.Furthermore,si RNA-mediated ALIX and TSG101 silence experiments revealed that NDV infection can promote the release of the exosome through ESCRT pathway,and ALIX silence can reverse the NDV induced ALIX expression increase.In the meanwhile,ALIX/TSG101 silence can also reverse the increase of the secretion of exosme caused by NDV infection.The effect of exosme on viral replication was also investigated.It showed that NDV Ex was not infectious,but the addition of extra NDV Ex and the treatment with exosome inhibitor(GW4869)showed that NDV Ex can promote NDV replication.Further investigation indicated that NDV transmitted NDV structural protein NP could be transferred to DF-1 cells through the exosme.The NP protein in the cell shows a promotive effect of viral replication and an inhibition ability of anti-viral cytokines.In general,the NDV Ex can inhibit the secretion of antiviral related cytokines by transferring the virus NP protein,and therefore promote NDV infection.Although transcription analysis showed that although most genes decreased significantly in the exosme after NDV infection,there was still a significant number of antiviral related genes increased after NDV infection.The secretion of these antiviral related factors may be antagonistic to the virus-promoting effect mediated by the exosme delivered NP protein.To clarify the role of these exosme genes in NDV infection,the intracellular expression levels of several antiviral related gene m RNA were examined.It was found that the change of NLRX1 before and after NDV infection were significant.Virulent NDV strains of infection not only promoted the content of NLRX1 m RNA in extracellular secretion,it also reduces the expression of NLRX1 protein and m RNA levels intracellularly.Moreover,treatment of GW4869 inhibitors can increase the NLRX1 content in cells while reducing the external secretion NLRX1 m RNA,suggesting that NDV infection can be excreted outward through the external secretion NLRX1 m RNA reduces intracellular NLRX1 content to promote the replication and proliferation of viruses.Further studies have shown that the exosome does not contain NLRX1 protein,and the NLRX1 m RNA excreted by the exosome will not be translated into NLRX1 protein in the infected cells,so it will not counteract the virus-promoting effect caused by NLRX1 m RNA discharge.In general,we obtained NDV Ex,clarified the pattern of NDV infection to promote the release of exosme.For the first time,it was revealed that the mechanism of exome to promote NDV infection and replication by delivering viral NP protein and host NLRX1 molecule,enriched the knowledge of exosome mediated NDV pathogenesis mechanism.It provides a novel theoretical basis for the prevention and control strategy of ND.