Study On The Mechanism Of NS4A Protein Of Duck Tembusu Virus Inhibits Interferon-β And The Attenuated Virus On Chicken Embryos

The outbreak of duck Tembusu virus infection（DTBSV）has spread around the major duck-producing regions in China since April 2010.It is characterized by severe egg drop syndrome and neurological symptom in domestic ducks,which causes a heavy hit on duck-producing industry.Duck Tembusu virus,a positive-sense,single-stranded RNA virus,belongs to Ntaya virus group within the genus Flavivirus,family Flaviviridae.As a newly emerged zoonotic disease,currently there are no commercial vaccines available for widely clinical use,or no effective drugs to cure this virus infection.Therefore,it is of significant importance to look into the pathogenic mechanisms to develop effective vaccines and antiviral drugs specific for DTMUV.In this research,a duck Tembusu virus strain named as XN virus which was isolated from a DTBSV-infected duck in Xianning,Hubei Province,was used for our study.The m RNA levels of adaptor molecules in the IFN-βsignal pathways and related cytokines were detected by real-time RT-PCR after duck embryo fibroblast（DEF）cells were infected with XN virus.Results showed that IFN-βwas inhibited during the early stage of virus proliferation,but downstream ISGs were not affected,which suggested the upstream signaling of IFN-βcould have been blocked.The RNA of XN virus was extracted and then reverse-transcribed into c DNA as the template for PCR.The effects of NS genes on the IFN-βpromoter activity and IFN-βtranscription level were assayed,and NS4A gene had a significant and stable inhibiting effect among the 7 genes.It indicated that NS4A could have a relationship with IFN-βinhibiting.IFN-β-induced IRF-3 and its upstream adaptor molecules were inhibited by NS4A in the promoter activity assays as well.NS4A can also suppress the protein expression of IRF-3,and suppress IRF-3-P accordingly,but it did not affect the m RNA levels of IRF-3.When added MG132（a proteasome inhibitor）,the inhibiting effect is attenuated,indicating that NS4A degrades IRF-3 protein through a proteasome pathway.NS4A was observed to be able to co-located with IRF-3 or IRF-3-P by confocal observation,furthermore,it could coprecipitate tightly with IRF-3 and slightly with phosphorylated IRF-3,suggesting that NS4A can degrade IRF-3 protein by direct interaction.Futher study indicated that NS4A cannot degrade IRF-3 protein through RBCK1 or TRIM pathway,it is speculated that NS4A might induce IRF-3 degradation through its own enzyme-like activy.In addition,in this research,XN virus was inoculated into the allantoic cavity of8-day-old embryonated chicken eggs and serially passaged.After harvesting the allantoic fluid,the virus is detected by RT-PCR using specific primers against the E gene.As a result,the expected band was observed after DNA electrophoresis.As the passages increased,XN virus propagated effectively and kept stable,and the embryos died collectively during 48hpi and 72hpi after 25 times.EID₅₀values were tested for the passaged virus,and reached a relatively stable virus titer.In the meanwhile,infected embryos exhibited characteristic pathological changes.Results indicated that the XN virus could adapt chicken embryos well.Genome of the adapted virus was sequenced to supervise the sequence mutation status.The results of animals experiment suggested that the proliferation ability of adapted virus-F80 in ducklings was attenuated,and the ability to lead lesions in tissues was attenuated,too.Results indicated that the virulence of the adapted virus-F80 on ducklings had decreased,and it was attenuated to a certain extent.