The Studies Of The Biological Properties And The Genetic Characteristics Of Quasispecies Of Diverse Large Palque And Small Plaque Of XZ2012 Strain Of The Duck Tembusu Virus

Duck Tembusu virus disease(DTMUVD),also called Duck egg drop syndrome,is a new type of acute infectious disease caused by The Duck Tembusu virus that mainly causes egg-laying ducks egg production drop and neurological symptoms on egg-laying ducks in later periods.In addition to infected ducks,DTMUV also can infect geese,chickens,pigeons,sparrows.DTMUV as a member of mosquito-borne virus cluster of flaviviruses,mosquitoes might be involved in the spread of this virus,also probable horizontal transmission of ingestion or inhalation of feces-contaminated material.The disease spread quickly and impact a wide range.Egg-laying ducks infected DTMUV,then the egg production rate can lay eggs peak(70%?90%)down to close to 10%.Duck Tembusu virus disease outbreak firstly on April of 2010 in a series of farms in southeast China.The disease quickly spread followed in Fujian,Shandong,Zhejiang,Jiangsu provinces,Guangxi Zhuang Autonomous Region,some of the largest density waterfowl breeding region as the disease popular widely.There were large outbreaks of 2011,2012 and 2014 years,now the disease has become Chinese poultry breeding industry a common disease,threating the development of the poultry breeding industry.At present,there was no commercial vaccine for the treatment of the disease,the farm should primarily improve aquaculture environment,good biosecurity prevention and control,appropriate to carry out drug prevention.The DMTUV is a single stranded and positive strand RNA virus,belonging to the Flavivirus genus,Ntaya virus group.WNV has been passaged 20 times in mosquitoes,DENV has been passaged 53 times in primary dog kidney(PDK)cells and DTMUV has been passaged 3 times in brains of BALB/c mices,there were the mix-plaque containing large plaque and small plaque,suggesting mutations.In this study,DTMUV XZ2012 strain was adapted to the BHK-21 cells for several generations and present the mix-plaque.After picking the single plaque,the diverse large plaque and small plaque quasisipecies were gene sequenced and study their biological properties.The thesis contains two parts:1.The study of the genetic characteristics of diverse quasispecies of diverse large plaque and small plaque of XZ2012 strain of the Duck Tembusu virusIn our laboratory,there was an adapted BHK-21 cell DTMUV XZ2012 strain isolated from Xuzhou area in Jiangsu Province in 2012 year.In the plaque formation experiment,it was found that the XZ2012 plaque phenotype was mixed with large plaques and small plaques indicating there may be more than one quasispecies.Picking the single plaque with pipettes tips three times,obtained 7 strains stable genetic plaque phenotype on Baby hamster kidney 21 cells.There were 3 strains of large plaque named as DTMUV JS-L1/2015,DTMUV JS-L2/2015,DTMUV JS-L3/2015 and 4 strains of small plaque named as DTMUV JS-S1,DTMUV JS-S2/2015,DTMUV JS-S3/2015,DTMUV JS-S4/2015.Designing 14 pairs of primers based on the XZ2012 gene sequences relesaed on NCBI(GenBank accession no:KM188953.1)to amplify the complete genomic sequences of all the 7 strains isolated clones.The results of the sequence were compared by MegAlign after NCBI.The results showed that the similarity with DTMUV XZ2012 was 99%.However,the differ between the large plaque strain and the small plaque strain was mainly reflected on the XZ2012 nucleotide position 1140 and 10699.The nucleotide position 1140 in E(envelope protein)gene of the XZ2012 strain of DTMUV had mutanted accounting for the amino acid E?A on amino acid position 62 of E protein,which can explain why the two quasispecies of DTMUV have different plaque phenotype on BHK-21 cell.2.The study of the biological characteristics of the large plaque quasispecies LI and the small plaque quasispecies S1 of XZ2012 strain of the Duck Tembusu virusEvaluating the DTMUV large plaque quasispecies L1 and DTMUV small plaque quasispecies S1 on BHK-21 cells,Vero cells and DF-1 cells indecited the plaque phenotype of the two strains was existed stably.In order to find the difference between the large plaque quasispecies L1 and the small plaque quasispecies S1 of DTMUV,the virus growth curve experiment was carried out on BHK-21 cells and C6/36 cells.The results showed that the virus content of L1 strain higher than the virus content of S1 strain whether the cell lysate or not the supernatant on BHK-21 cell.In general,there were no significant differences between the L1 strain and the S1 strain in C6/36 cell.But the growth rate of small plaque quasispecies S1 is faster than the growth rate of large quasispecies plaque L1 in the early stage and the time of CPE appearance of S1 is earlier than L1 one day.In order to compare the pathogenicity of the large plaque clone with the pathogenicity of the small plaque clone,the S1,L1 virus solution(PFU=7x105/mL)were used to determinate the ELD50 on SPF duck embryos.The results showed that the ELD50 of S1 was 10-4.64/0.1mL,less than the ELD50 of L1 10-3.88/0.1mL,indicating that the toxicity of the small plaque quasispecies S1 is stronger than that of big plaque quasispecies L1.In order to analyze the difference in antigenicity,the L1 and S1 viruses solution were inactivated mix with the adjuvant ISA-206 immune mice and detect the antibody level differ between the L1 and the S1.The result indicted that the antigenicity of the small plaque quasispecies S1 was stronger than the antigenicity of the large plaque quasispecies LI,the S1 can induced higher antibody level at equal dose.