| Duck Tembusu virus(DTMUV)appeared in China in 2010,caused an avian infectious disease characterized by feeding,egg descent and paralysis,which is called duck Tembusu virus disease.DTMUV was mainly harmful to ducks and geese,and there were also reports of chicken infection.The homology of the isolates was higher than 98%.The incidence rate of the disease is high in season which mosquito is active,but duck Tembusu virus disease is also occrus in winter.So the transmission of duck Tembusu virus is not limited to mosquitoes.Duck Tembusu virus belongs to Tembusu virus,Ntaya virus group,flavivirus,Flaviviridae.The genome of duck Tembusu virus is liner single-strand RNA,the length is llknt.Genome structure is 5’UTR-C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3’UTR.The envelope protein(E protein)is the major structural protein of DTMUV,which is involved in the binding of virus and host cell receptors.E protein contains the main neutralizing epitopes of the virus.NS1 protein of DTMUV is also involved in the assembly of the virus,and it is secreted to the extracellular in the form of the two monomer or the dimer.It is antagonistic to the innate immunity of the host.E protein and NS1 protein are major protective antigens of DTMUV,and the information of their epitopes is less.In this study we isolated and identified a strain of duck Tembusu virus.We studied its biological characteristics,determined its total gene sequences,and analyzed its gene evolution features.Cell fusion technique was used to prepare 4 strains of anti-E protein and anti-NS1 protein monoclonal antibodies(McAb).The monoclonal antibodies against E protein can distinguish between DTMUV and Japanese encephalitis virus(JEV)antigen.It could lay the foundation for the establishment of DTMUV serological diagnosis method.Specific studies are as follows:1.Isolation and gene variation character of duck Tembusu virus strain XZ-2012In October 2012,an infectious disease of cherry valley breeder ducks were happened in a duck farm located in Xuzhou,Jiangsu provine.The affected ducks showed a series symptom of febrile,severe loss of appetite,drop in egg production,paralysis and even death.Post-mortem examinations revealed swollen spleen and severe hemorrhagic ovaritis.We isolated the causative agent,a duck tembusu virus,from affected ducks and designated it XZ-2012 after determined the partial genome sequence.No hemagglutination activity was observed with the isolated virus in allantoic fluid.The DTMUV strain XZ-2012 can adapt to BHK cells and the virus titer of the 5th generation is 5×104PFU/mL.The genomes of DTMUV strain XZ-2012 is 10,990 nucleotides(nt)in length and encode a putative polyprotein of 3426 amino acids.It is flanked by a 5’ and a 3’ noncoding region(NCR)of 94 and 618 nt,respectively.The whole genome homology between XZ-2012 and previous reported virus isolates BYD,YY5 and JS 2010 et al.was 98.5%to 99.3%.Nucleotides mutation of DTMUV XZ-2012 strain are mainly located in E,NS1 and NS2 protein encoding genes,which indicates that corrently transmitted DTMUV may endure immune pressure in nature.Information of the whole sequence of XZ-2012 strain will be useful for vaccine candidate selection and further investigation of the molecular epidemiology of DTMUV in china.2.Development and Identification of monoclonal antibody against EDⅢ protein of duck Tembusu virusIn the present study,the purified EDⅢ protein was used to immunize BALB/c mice.The stimulated splenocytes were fiised with myeloma cells of SP2/0 to produce hybridomas.Two stable murine monoclonal cell lines producing antibodies(McAbs)against EDⅢprotein were generated and named 3C4 and 3D12.The titers of the two McAbs in the supernatant of cell culture were 1:12800 and 1:12800 and the titers in ascites were1×105 and 2×105 respectively.Subclass identification results showed that 3C4 belonged to IgGl subclass,while 3D12 were found to be of IgG2b subclass.Both of the McAbs had κ light chain.Western blot and IFA analysis indicated that 3C4 and 3D12 could react with EDIII protein of DTMUV,while have no cross reaction with JEV.Neutralization test showed that the two mAbs had no neutralizing activity.3.Development and Identification of monoclonal antibody against NS1 protein of duck Tembusu virusFor preparing monoclonal antibodies against NS1 protein of DTMUV,BALB/c mice were immunized with the purified NS1 protein,and 2 stable hybridomas were produced,named as 2F6 and 2F12.The titers of the two McAbs in the supernatant of cell culture were 1:6400 and 1:12800 and the titers in ascites were both 1×105 respectively.Subclass identification results showed that 2F6 and 2F12 belonged to IgGl subclass.Both of the McAbs had κ light chain.Western blot and IFA analysis indicated that 2F6 and 2F12 could react with NS1 protein of DTMUV.While 2F6 has no cross reaction with JEV,2F12 has cross reactions with JEV.4.B cell epitope identification of duck Tembusu virus EDⅢ proteinIn this study,truncated EDⅢ protein were expressed successfully as GST-fusion protein in E.coli.system and peptides were synthetized to determine the epitopes of the monoclonal antibodies(McAbs)against the DTMUV EDⅢ.Through the identification of ELISA and western blot,Epitope mapping results indicated that the two McAbs specifically recognized the epitope 310SLVKNP 315.This epitope was found on nalysis to a highly conserved,and do not cross with other Flaviviridae virus.All the results might promote the future investigations of the antigen structure and function of EDIII of DTMUV and facilitate the identification and development of diagnostic methods for DTMUV infection. |
