Effect Of Diffrent Ed? To The Infectious Of Tembusu Virus FX2010 Straiin And RMM1775 Strain

Since the outbreak of the Duck Tembusu virus(TMUV)disease from 2010 in China,to the duck breeding industries.The disease was mainly characterized by the drop of egg production,high fever,loss of appetite,dyskinesia and may cause the death of ill ducks.TMUV FX2010 strain(FX2010)was successfully isolated and the biological characteristics of FX2010 were studied previously.In 1955,the first TMUV strain MM1775 was isolated from mosquito in Malaysia.Previous studies showed that the replication,pathogenicity and contact transmissibility of FX2010 and rMM1775 differed in ducks,but the molecular mechanism was not clear.TUMV envelope(E)protein can be divided into three domains,Domain I,Domain? and Domain?.The domain III mainly mediate the combination of virus and host cell receptor protein,which may be closely related to the pathogenicity of the virus.By comparing the amino acid residues of E domain? of FX2010 and MM1775,It was found that the differences were located in E312 and E332,respectively.To study the potential difference of virus pathogenicity caused by these two amino acid residue changes,two plasmids FX2010-E/V312A/T332S and MM1775-E/A312V/S332T were engineered by using direct-site mutagenesis PCR,fusion PCR and cloning.Then the full length viral cDNAs,with T7 RNA polymerase promoter sequence added at the 5 'terminus,were amplified by using long fusion PCR,respectively.The cDNAs were used as templates to transcribe RNAs in vitro,respectively.The infectious RNAs were transfected into fresh DF-1 cells,respectively,and the specific cytopathic effects(CPEs)appeared 48 hours post transfection.The supernatants were collected and infected into fresh DF-1 cells when 90%transfected cells showed CPEs.Apparent CPEs were observed 72h.p.i.Specific green fluorescence in the newly infected cells could be detected by IFA test.Ten segments of cDNA,overlapping at 5 ' and 3 ' terminus,containing the whole genome of virus,were amplified by RT-PCR.Sequence analysis confiremed the rescued viruses with the expected nucleotide change,which meant the successful rescue of rFX2010-MM1775-EDIII and rMM1775-FX2010-EDIII,respectively.To study the infection of mutant viruses in ducks,six 12-week old ducks were inoculated with 103.5TCID50 of rFX2010-MM1775-EDIII or rMM1775-FX2010-ED?,respectively,and three naive ducks were introduced into the isolator,in which the inoculated ducks were housed.At 4 day p.i.,the inoculated ducks were euthanized and the tissue samples were collected and titrated.The result showd that rFX2010-MM1775?EDIII was mainly detected in spleen,spleen,lung,kidney,ovary and brain of infected ducks.Sera antibodies against TMUV of three contact ducks were positive at 7,14 day p.c.,which meant that rFX2010-MM1775-EDIII could transmit from infected ducks to contact ducks.rMM1775-FX2010-EDIII was mainly detected in spleen and liver of inoculated ducks.Sera antibodies against TMUV of three contact ducks were negative at 7,14 day,which meant that rMM1775-FX2010-EDIII could not transmit from infected ducks to contact ducks.It is inferred that the ED III is not the main regne that controls the ability to propagate or is able to control its communication ability with a certain region,it still need to study in experiment in the future.