Stem-Loop-I Structure Mapped In Tembusu Virus 3’ Untranslated Region Regulates Viral In Vitro Proliferation And Mice Pathogenicity

Tembusu virus(TMUV)is a mosquito-borne flavivirus,that mainly infects avians.In1955,TMUV was first isolated from Culex tritaeniorhynchus in Malaysia.Since 2010,TMUV has broken out and spread rapidly in the duck farms in the southeast coastal area of China,which caused huge economic losses to the duck industry.Besides,mice can be infected with TMUV artificially,and different TMUV strains possess different pathogenicity in mice.It’s important to understand the molecular mechanisms of TMUV interspecies transmission and the pathogenicity differences in mice for preventing the spread of the virus.Many studies showed that the repeat secondary structures located at the 3’ untranslated region(3’UTR)of mosquito-borne flaviviruses faced different host selection pressures in the process of virus adaptation to different hosts,and different mosquito-borne flaviviruses had different evolutionary strategies for adaptation to different hosts.What’s more,the 3 ’UTR of mosquito-borne flavivirus was also closely related to viral virulence.To determine the effects of the TMUV 3′-untranslated region(3′UTR)in viral host-specific adaptation and in the pathogenicity for mice,we generated a set of chimeric viruses using CQW1(duck strain)and MM＿1775(mosquito strain)as backbones with heterogeneous 3′UTRs.Compared with r MM＿1775,r MM-CQ3′UTR(recombinant MM＿1775 virus carrying the 3′UTR of CQW1)exhibited enhanced proliferation ability in vitro,with peak titers increasing by 5-fold in duck embryonic fibroblast(DEF)cells or 12-fold in baby hamster kidney(BHK-21)cells.However,the neurovirulence of r MM-CQ3′UTR was attenuated in 14-day-old Kunming mice via intracranial injection,showing slower weight loss,lower mortality,and reduced viral loads in the brain.In contrast,r CQ-MM3′UTR(recombinant CQW1 virus carrying the3′UTR of MM＿1775)showed similar growth kinetics in vitro and neurovirulence in mice compared with those of r CQW1.Then,the Stem-loop-I(SLI)structure,which showed the highest variation within the 3′UTR between CQW1 and MM＿1775,was further chosen for making three recombinant viruses(r MM-CQ3′UTRSLI,r CQ-MM3′UTRSLI and r CQ-3′UTRSLIΔ68).The in vitro characteristics of recombinant SLI viruses showed that while the peak titers of r MM-CQ3′UTRSLI displayed a 15-or 4-fold higher in DEF or BHK-21 cells than r MM＿1775,r CQ-MM3′UTRSLI and r CQ-3′UTRSLI Δ 68 showed similar multiplication ability compared with r CQW1.Meanwhile,the pathogenicity of the mice infected with the three SLI recombinant viruses was attenuated than that of the parental viruses.To further validated viral host-specific adaptation function of the TMUV SLI structure,we further constructed and rescued the SLI deleted reporter virus using a DNAbased CQW1 reporter virus(CQW1-Nluc)as the backbone.We found that,compared with the Nluc-WT,the SLI-deletion mutant reporter virus(CQW1Nluc-3′UTRΔSLI)showed comparable growth kinetics in C6/36 cells but attenuated growth ability both in DEF and BHK-21 cells.In conclusion,we demonstrated that the SLI structure in the TMUV 3′UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old Kunming mice,providing a new understanding of the functions of TMUV 3′UTR in viral multi-host fitness.