Attenuation Strategy Of Duck Tembusu Virus Based On Dysfunction Of C Protein

Tembusu virus(TMUV)is an emerging avian flavivirus,causing oophoritis and encephalitis resulted in egg-drop syndrome,mainly in ducks and geese.Since the first outbreak in southeastern coastal areas of China in 2010,TMUV has become one of the major pathogens in waterfowl industry of China,and is epidemic in the certain regions.Although there are several commercial vaccines for TMUV,and significantly decreased the morbidity,in order to counter the new epidemics and threats caused by viral variation,it is important to study and develop new vaccines for TMUV.In the present study,we successfully constructed a full-length cDNA clone for a clinical TMUV strain CQW1 via subclone,and recovered infectious recombinant virus which’s genome sequence was identical to the parental virus,except for the engineered genetic marker.Besides,the recombinant virus showed comparative growth kinetics and virulence to parental virus.Based on the reverse genetics system,we further successfully generated a stale reporter virus which carrying a Nano Luc gene.We also constructed subgenomic replicons and a packaging system for TMUV.These reverse genetics technology-based multicomponent tools,established an integrated platform for studying each step of viral life cycle,and are beneficial to the study of viral replication mechanisms and development of new vaccines.Flavivirus C protein(CP)plays an essential role on viral assembly process.The predicted structure of TMUV CP reveals that it’s constituted by four distinct α-helix.Based on this structure,we screened the structural requirements of TMUV CP for viral morphogenesis by introducing a series of internal mutations using reverse genetics technology in combination with replicon packaging assays.TMUV CP showed a dramatic functional and structural flexibility,even though 44 residues at N-terminus were deleted,it was still capable of packaging replicon RNA;in addition,33 residues were removed from the C-terminus(containing α3 and nearly the entire α4-helix),and infectious particles were still produced.A central goal of the present study is to assess whether CP could act as a target to attenuate TMUV and be used for live attenuated vaccine(LAV)development.We further analyzed two mutants(ΔC20-43 virus and ΔC64-96 virus)with relatively large deletions.Both ΔC viruses showed attenuated virus proliferation in cells and attenuated virulence in duck embryos;impaired viral RNA replication and assembly are the molecular basis for the attenuated phenotypes of ΔC20-43 virus and ΔC64-96 virus,respectively.Although continuous passages resulted in better growth phenotypes,these deletion mutations are quite stable in cell culture.Animal assays indicated that both ΔC20-43 virus and ΔC64-96 virus were highly attenuated in ducklings but still immunogenic.Single-dose immunization with any of ΔC20-43 virus or ΔC64-96 virus could protect ducklings from a lethal challenge.And our data shed light on replication/assembly defective TMUV with internal deletions in CP is an effective approach for developing LAV.Next,we revealed that TMUV C-prM polyprotein junction was sequentially cleaved by NS2B/3 and signalase,unlocking this coordinated process resulted in viral assembly defect.In order to find a better method to develop LAV,using TMUV as a flavivirus model,we generated two bicistronic viral genomes—CQW1-IRES-mC and CQW1-MINI-mC,which ectopically expressed CP under the control of internal ribosome entry site,in their viral3’UTRs.The independent expression of CP brakes spatially and temporally regulated C-prM cleavage,resulting in less efficiency of recruitment of CP to viral assembly site.Both CQW1-IRES-mC and CQW1-MINI-mC viruses are highly attenuated in vitro,due to the defected viral assembly,but both showed a good genetic stability.Animal experiments indicated that both CQW1-IRES-mC and CQW1-MINI-mC viruses were highly attenuated in mice and ducklings,did not causing any obvious symptoms.But a single-dose of immunization with either viruses could efficiently active ducks’ cellular and humoral immunity,and protect ducklings from a lethal challenge and viremia.Besides,compared with ΔC-virus strategy,this method is more universal in Flavivirus genus.Altogether,we established a powerful reverse genetics platform for TMUV,and revealed that the structure of CP of TMUV is not precisely corelated with its functions in viral morphogenesis.Dysfunction of CP would result in defect of viral replication or assembly process,and making CP can serve as a target to attenuate TMUV.The present study also firstly demonstrated that ectopically expresses structural proteins could be an effective and universal method for flavivirus LAV development.These data would contribute to the development of new types of flavivirus vaccines.