Development Of Mutational Duck Tembusu Virus Based On The Cap-independent Translation Pathway

Duck Tembusu virus(DTMUV)is a newly emerged causative agent of duck egg drop syndrome classed into Flavivirus genus,Flaviviride family.Protein synthesis plays key role in virus proliferation and study on the translation mechanism of DTMUV is an important approach to elucidate its pathogenic mechanism and research on prevention and treatment.We constructed DTMUV translation template by using renilla luciferase gene replace ORF of DTMUV.Based on reporter m RNA assay,we first confirmed that DTMUV UTRs can initiate the cap-independent translation in an IRES-independent manner and highly depend on 5' end.The cap-independent translation was also observed in other flavivirus in different cells and showed host selectivity.By replacing,deletion and mutation in UTRs of the translation template,we confirmed that primary and higher structures of DTMUV UTRs are important for its efficient cap-independent translation.Based on reverse genetic system of DTMUV,we determined the effect of inhibition of cap-independent translation of DTMUV for virus proliferation.We constructed mutational replicons which are mutation in DB2,PK2 and SL regions 3'UTR of DTMUV,mutation inhibiting cap-independent translation decreased DTMUV proliferation in vitro and delayed,but did not prevent,death of infected duck embryos.Finally,our study obtained a mutational duck Tembusu virus,but the pathogenicity and potential for vaccine were not further study due to the no significant attenuation of pathogenicity of mutational recombinant virus in vitro.