The Role Of Glycosylation Of Duck Tembusu Virus E Protein In Viral Proliferation And Pathogenesis

Duck Tembusu virus disease is an acute,contact new infectious disease caused by Duck Tembusu virus?DTMUV?.In 2010,DTMUV broke out for the first time in East China,and quickly spread to poultry farms in more than ten provinces and cities nationwide.The disease mainly causes the laying of the laying ducks to drop or even stop.In the meat ducks,the symptoms are mainly viral encephalitis,the incidence rate is over 90%,and the mortality rate is 5-30%,which brings serious economic losses to the duck industry in China.The glycosylation modification of flavivirus E protein can affect viral replication,cross-species transmission and pathogenicity.However,the role of DTMUV E protein N154 glycosylation modification in viral replication,pathogenesis and host encephalitis is not fully understood.This study established the reverse genetic system of DTMUV,constructed mutant viruses of DTMUV E protein N154 glycosylation site,and analyzed the N154glycosylation of E protein in DTMUV replication,pathogenicity and viral encephalitis formation.To lay the foundation for studying the molecular mechanism of DTMUV pathogenesis and the development of new vaccines.The main contents are as follows:1.The role of E protein glycosylation in DTMUV replicationThe growth curves of DTMUV wild-type virus and E-protein glycosylation mutant virus on DEF and C6/36 cells were determined by TCID₅₀ assay,and it was found that the titer of E-protein glycosylation mutant virus was significantly lower in DEF and C6/36 cells.Further studies have found that depletion of E protein glycosylation reduces the adsorb and invade of DTMUV,and the efficiency of DTMUV replication,assembly and release in DEF and C6/36 cells.2.The role of E protein glycosylation in DTMUV-induced inflammatory response in duck glial cellsInfecting duck glial cells with DTMUV E protein glycosylation mutant virus and wild type virus,respectively.By relative qRT-PCR fluorescence quantitative experiments,it was found that depletion of E protein glycosylation attenuated the expression levels of DTMUV-induced pro-inflammatory cytokines TNF-?,IL-1?,IL-6,IL-12 and chemokines IL-8 and CCL5.It is suggested that the elimination of E protein glycosylation attenuates the inflammatory response induced by DTMUV in duck glial cells.3.The role of E protein glycosylation in the pathogenicity of DTMUVThe duck challenge test found that depletion of E protein glycosylation increased the weight gain of DTMUV-infected ducks,while clinical symptoms,mortality,viremia,and viral load in brain,spleen,liver,and kidney were significantly reduced.It is suggested that E protein glycosylation plays an important role in DTMUV pathogenesis.4.The role of E protein glycosylation in DTMUV-induced neurovirulenceThe challenge test found that depletion of E protein glycosylation significantly reduced the vascular cuff,satellite phenomenon,phagocytic phenomenon,glial cell nodules,inflammatory cell infiltration and meningeal thickening of ducks infected with DTMUV.Immunohistochemical observations revealed that elimination of E protein glycosylation significantly reduced the loading of DTMUV in brain tissue.It indicates that glycosylation of E protein plays an important role in the neurovirulence caused by DTMUV.5.The role of E protein glycosylation in DTMUV-induced inflammatory response in duck brainThe cerebral cortex of each group ducklings was stripped.By relative qRT-PCR fluorescence quantitative experiments,it was found that depletion of E protein glycosylation attenuated the expression levels of DTMUV-induced pro-inflammatory cytokines TNF-?,IL-1?,IL-6,IL-12 and chemokines IL-8 and CCL5.It is suggested that the elimination of E protein glycosylation attenuates the inflammatory response induced by DTMUV in duck brain.Taken together,this study firstly reveals the effect of E protein glycosylation on DTMUV replication cycle,pathogenicity,neurotoxicity,and host neuroinflammatory response.It is great significance for us to understand the pathogenesis of DTMUV more deeply,and it also provides new ideas for vaccine design and drug screening for anti-DTMUV.