The Evolution And Molecular Basis Of Attenuation Of Duck Tembusu Virus

Since 2010 duck Tembusu virus?DTMUV?disease firstly broken out in China,there have been constant reports about infection.13 strains of DTMUV,subsequently named ZJ201501,ZJ201502,ZJ201503,ZJ201504,ZJ201505,ZJ201506,ZJ201507,ZJ201508,JS201501,JS201502,AH201501,AH201502 and AH201504,were isolated successfully on DF-1 cell and identified by RT-PCR,which derived from sick shelducks'samples in Zhejiang,Jiangsu and Anhui provinces in 2015.Each of 9overlapping segments covering the whole genomes of these isolates was amplified by PCR,then the PCR products were sequenced and assembled using Seqman software to get the complete sequence of each virus.The phylogenetic trees were drawn based on the ORFs and the E proteins,respectively,using MegAlign and Mega7.0 software,compared with the sequences of 15 DTMUVs from our laboratory and in GenBank from 32 DTMUVs in different years.The isolates from 2015 are always in a independent branch and on the top in the phylogenetic trees on the basis of ORFs or E proteins,showing a near genetic distance between GX2013G,GX2013C,CQW1 and SD201120 but a far genetic distance from earlier strain FX2010.They had more than 97.1%nucleotide identities and more than 98.4%amino acid identities based on E proteins,indicating that DTMUV showed a certain degree of mutation in ducks in the past years.However,the sequences of ORFs and E protein amino acid are still kept in a high homology.In order to study whether the pathogenicity has changed,an experiment on 33-week shelducks was conducted by i.n with a dose of 10^3.5TCID₅₀ challenged viruses?SH201002,JS201101,HB201214,ZJ201503 and ZJ201502?chosen by the location of evolutionary tree and the time in different years.Almost all infected ducks present significant clinical symptoms,including inappetence,gross and microscopic lesions,especially severe swelling spleen and serious ovary hemorrhage.The viral isolation indicates all the viruses can replicate in the lung except SH201002,and the result of other viral isolation was slightly different which indicates the tissue tropism of DTMUV may have gone through a dedicate change.All most of the ducks were still alive but JS201101 when two ducks were dead at 8dpi and 12dpi,respectively,the time of seroconvesion of SH201002 was later than other viruses,demonstrating the antigenicity in ducks changed.An experiment on 3-week BALB/c mice was conducted by i.n with a dose of 10^4.0 TCID₅₀ of 13strains of isolates to explore the pathogenicity in mammals.There was no any obvious symptom in mice and evident decrease in weight change compared with PBS negative group just with a certain of inhibition,while some viruses can be isolated from nasal and lung samples manifesting DTMUV has acquired the ability to replicate in mammals.To deeply figure out the molecular basis of virulence of DTMUV,FX2010 and attenuated FX2010-180P?180P?were used as the experimental subjects.On the basis of reverse genetic operating system by replacing the E gene alternatively,2 recombinant viruses of r180P-FX2010E and rFX2010-180PE?our lab has constructed?were needed to be rescued,then an experiment on 9-week shelducks was carried out by i.m with a dose of 10^3.5TCID₅₀of rFX2010,r180P,rFX2010-180PE and r180P-FX2010E.Comparing with rFX2010,rFX2010-180PE can still present system infection but lost the ability of horizontal transmission through direct contact.The virulence of r180P-FX2010E hasn't enhanced and the virus can merely copy at low level in spleens and can't transmit among the ducks.To identify the key amino acid affecting the horizontal transmission,3 domain recombinant viruses of rFX2010-EDI?rFX2010-EDII and rFX2010-EDIII which each domain contains different amino acid sites compared with 180P were rescued at the background of FX2010,subsequently were used to do a research on shelducks.The virulence of rFX2010-EDI and rFX2010-EDIII hasn't changed a little,however,the virulence of rFX2010-EDII has markedly decreased demonstrating low copies in spleens and no horizontal transmission any longer.rFX2010-E/E89G and rFX2010-E/D120N consist of 2 amino acid difference in domain II were proceeded to be rescued and worked to research on ducks.Results found rFX2010-E/E89G had the same characteristics with the parent strain of rFX2010,while rFX2010-E/D120N lost the strong pathogenicity which only could copy at a low amount in spleens and had no capacity to transmit any more demonstrating the same biologic characteristics of 180P.This reveals the mutation of E120 site from D to N can dramatically reduce the viral replication and the ability to horizontal transmission which is important for the attenuation of virulence.This research laid a good foundation for further study the molecular mechanism of pathogenisis of DTMUV and other flaviviruses.