Study On The Mechanism Of Targeted Attenuation For Duck Tembusu Virus Based On Viral Methyltransferase K-D-K-E Catalytic Tetrad

Duck Tembusu virus(DTMUV)is one of the main pathogens threatening duck industry in China.It belongs to family flaviridae,genus flaviridae.5’ cap methylation of flavivirus genome is an important step in virus proliferation.N-7 and 2’-O methylations are necessary for the formation of the 5’ cap structure of flavivirus genome.Flaviviruses have evolved itself methyltransferase(MTase)to perform N-7 and 2’-O methylations of their RNA cap.MTase located in the N terminal of NS5 protein has both N-7 and 2’-O MTase activities and contains the conserved K61-D146-K182-E218 catalytic tetrad sites.At present,many studies have shown that the entire K61-D146-K182-E218 motif is essential for 2’-O MTase activity,whereas N-7 MTase activity requires only D146.In this study,we investigated the effects of DTMUV K61-D146-K182-E218 on viral proliferation,virulence,translation,and host innate immunity,and understood the function of 5’ cap methylation of virus genome and its role in virus pathogenesis.Finally,the feasibility of the K61-D146-K182-E218 point mutated DTMUV as candidate of live attenuated vaccine was evaluated.1.Effects of K-D-K-E point mutation on the biological characteristics of DTMUVTo study the effects of K61-D146-K182-E218 point mutation on DTMUV biological characteristics,the replicons containing K61A,D146A,K182 A or E218A point mutation were successfully constructed using a DTMUV replicon operating system.The activities and copy numbers of the replicons showed that K61-D146-K182-E218 point mutation inhibited the replication capability of DTMUV.Based on DTMUV infectious cDNA clone,4 strain recombinant DTMUV(K61A,D146 A,K182A and E218A)were successfully saved.The genetic stability results showed that K61A,K182A and E218A mutants were genetically stable,while D146 A mutation was restored to D146 in the third passage.The growth curve of mutant viruses in duck embryo fibroblasts(DEFs)and its virulence in duck embryo were further measured.The results showed that the proliferation and virulence of K61A,K182A or E218A mutant virus were significantly reduced.2.K61A,K182A or E218A mutation affects DTMUV translation efficiencyTo explore whether the K61A,K182A or E218A mutation would affect the translation efficiency of DTMUV,we determined the viral titers,copy numbers,and protein expression of the recombinant viruses during protein expression period.The results showed that K61A,K182A or E218A mutation reduced the translation efficiency of DTMUV and thus reduced virus formation.To further explore which methylation defect(s)affect the translation of the virus,we obtained translation template RNA containing different cap structure components.It was found that deficient in N-7methylation inhibited translation of DTMUV,suggesting that the deficiency of N-7methylation was the key factor leading to the inhibition of DTMUV translation by K61-D146-K182-E218 point mutation.3.K61A,K182A or E218A mutant DTMUV induces innate immune responseTo explore the effect of K61A,K182A or E218 mutant DTMUV on innate immune response,we measured the transcription levels of cytokines using RT-qPCR after viral infection.The results showed that DTMUV with mutation at K61A,K182A or E218A could significantly induce host innate immune response compared to WT virus.To further understand the effect of mutant viruses on innate immune response,we selected E218A and WT DTMUV infected cells for transcriptome sequencing.The results showed that E218A mutant virus could significantly activate RIG-I-like receptor signaling.Further,RT-qPCR and dual luciferase reporting assay were used to identify the reliability of transcriptome data.4.Immunogenicity and immunoprotection of K61A,K182A or E218A mutant DTMUVDTMUV with mutation at K61A,K182A or E218A showed good attenuated characteristics in vitro.To investigate whether recombinant DTMUV K61A,K182A and E218A could be used as live attenuated vaccine candidate strains,the immunogenicity and immunoprotection of mutant viruses were determined in duckling.These three mutants were highly attenuated in both virulence and proliferation of the virus in ducklings but still immunogenic.Furthermore,a single-dose immunization with K61A,K182A or E218A could induce robust T cell responses and humoral immune responses,which could protect ducks from the challenge of DTMUV-CQW1.The results suggested that K61A,K182A and E218A mutated viruses have potential as live attenuated DTMUV vaccine candidate strains.In conclusion,we investigated the effects of K61-D146-K182-E218 mutation on the biological characteristics of DTMUV,further elucidated the reasons for the decreased proliferation and virulence of DTMUV with K61A,K182A or E218A mutation,and finally evaluated feasibility of K61A,K182A or E218A mutated DTMUV as live attenuated vaccine candidate strains.