The Discovery And Identification Of The Key Site Of Virulence In The E Protein Of Duck Tembusu Virus

Since April 2010, outbreaks of severe disease in ducks whose antigen is duck tembusu virus have widely spread around major duck-producing regions in China distributed in Zhejiang, Fujian, Guangdong, Guangxi, Anhui, Jiangsu, Henan, Shandong and Hebei provinces. Some symptoms of infected ducks are dgg-grop, high fever, diarrhea,anorexia,the infection rate in the ducks is 100% and the mortality of the diseased ducks is 10%~30%. The dsease caused enormous economic losses and has done harm to for public health. Duck Tembusu virus(DTMUV), a newly emerged duck virus that belongs to the family Flaviviridae, genus Flavivirus. The family of flaviviridae including Japanese encephalitis virus(JEV), Dengue virus(DENV), West Nile virus(WNV) and yellow fever virus(YFV) has posted a threat to global health security. However, recent research has determined that DTMUV isolated from geese, chickens, sparrows and mosquitoes has the ability of interspecies transmission from ducks to other animals.And in recent study the virus is lethal to mammals such as mice. So the disease may have interspecies tranmission from ducks to other mammals. However, no vaccine is currently available for prevention of the disease, we need take some measures to find the crucial site of virulence in E protein and reasearch the pathogenic mechanism and vaccine development.The content of this study inclueds the following four parts:1. The analysis and identify of the DTMUV virulence determinates sites.To research the virulence determinates sites of DTMUV, we obtained different DTMUV strains via the serial passages in mice brain. The different strains are isolated two strains named DTMUV-L and DTMUV-S through plaque purification technique.The two virus are different in the size of plaques and the neurovirulence. The results of sequence analysis indicated some significant mutation sites between these viruses.One of the mutations is belonging to E protein(serine become arginine) and others are belonging to the NS1 and NS5. Because the E protein is a major antigenic target of neutralizing antibodies and a receptor-binding to cells, so we establish research content and technical course on this mutated site.2. The construction of the S388 R mutant viurs of DTMUV To research the function of mutated site of DTMUV, we based on a infectious clones p CJDTMUV which are constructed with JEV strain E70 and pr M, E of DTMUV. Then induced the S388 R site into p S388 R and rescue the chimeric virus named r S388 R.3. The research of rescued virus about biological characteristics in vitro.The part is research on the virological characteristic between r S388 R and parental virus r WT in vitro. The results indicated that the r S388 R has some significant differences on growth cureve on Vero, DF-1 cells, the size of plaque, the temperature sensitive and the way of receptor-binding to cells. The r S388 R titer is much lower than r WT on Vero, DF-1 cells, the plaque get much smaller, the viral temperature stability get week, the tropism of receptor-binding is affected with HS. But there are no sigificant differences on growth curve on BHK-21, C6/36 and SH-SY5 Ycells and the viral protein expression.4.The analysis of neurovirulence, neuroinvasiveness, immunogenicity and immunoprection of the r S388 R.In animal experiments, we measure the neurovirulence, neuroinvasiveness,immunogenicity and immunoprotection of mutated virus in BALB/c and A129. The results showed that the r S388 R is obviously attenuated in neurovirulence and neuroinvasiveness. The r S388 R is also induced comparable humoral immunity as r WT and high titer of neutralizing antibodies. And the r S388 R can protect the mice away from the lethal dose of DTMUV challenge through subcutaneous immune reaction.Over all, we discovered a important mutated site in E protein via the serial passages in mice and constructed a chemeric infectous clone containing the mutated site.Through rescue the mutated virus and experiment on the molecular biology characteristic and pathogenesis mechaism. The research results indicated that the S388 R mutation is crusial for DTMUV, the site may play a imporatnt role in viral infection and provided a new strategies for vaccine developing.