Contribution Of Non-structural Proteins 3C And 3D On The Virulence Of Enterovirus 71

Enterovirus 71(EV71),a member in the family of Picornaviridae,can cause hand,foot and mouth disease(HFMD).It mainly infects young children and infants,especially children under 5,with typical clinical manifestations of fever,oral herpes,and rash on the hands and feet.However,HFMD may causesevere neurogenic complication,such as neurological pulmonary edema,aseptic meningitis and even death.So far pathogenic mechanism of EV71 remains unclear.Our earlier research indicated that there was difference replication capacity between different strains of EV71,and 3C and 3D proteins play important roles in replication of EV71.In this study,reverse genetics method was used to research roles of 3C and 3D regions in replication of EV71.Our study will provide theoretical basis for prevention and treatment of HFMD induced by EV71.ObjectiveTo explore roles of 3C and 3D proteins in replication and virulence of EV71,and this will be achieved by recombinant viruses SDLY107(1-3C)and SDLY107(1-3CD)which 3C and 3D regions of SDLY107 were replaced by that of SDLY1,respectively.Methods1.The replicon of pMD19-T-5'UTR-EGFP-3C-3'UTR was constructed by using technology of gene cloning.The recombinant 3C protein was observed by microscope.Growth curve of replicon was detected by Real-time quantitative PCR.2.Reverse genetics method was used to constructed recombinant viruses SDLY107(1-3C)and SDLY107(1-3CD)which 3C and 3CD regions of SDLY107 were replaced by thoseof SDLY1,respectively.After that,the viral titer was determined by TCID50 andtotal RNA was detected by qPCR.3.Replication and cell injury of recombinant viruses SDLY 107(1-3C)and SDLY107(1-3CD)were detected in vitro.Replication of EV71 was detected by qPCR and cell injury induced by EV71 was detected by LDH assay and CCK-8 assay.4.Replication and virulence of recombinant viruses SDLY107(1-3C)and SDLY107(1-3CD)were detected in vivo.Growth curves of EV71 in hindlimb muscle were detected by qPCR.Symptoms,weight and histopathological changes were observed to detected virulence of EV71 in ICR mice.Results1.The replicon of pMD19-T-5'UTR-EGFP-3C-3'UTR was constructed successfully.Although 3C protein expressed in RD cells,replicon could not replicate in RD cells.So,the replicon could not be used to research roles of proteins in replication of EV71.2.The recombinant viruses SDLY107(1-3C)and SDLY107(1-3CD)were constructed and rescued successfully.3.Replication of recombinant viruses SDLY107(1-3C)had no difference with that of SDLY107 at at 37? and 39.5?.No difference was observed between recombinant viruses SDLY107(1-3C)and SDLY107 in cell injury at 37? and 39.5?.Growth curve of recombinant viruses SDLY107(1-3CD)was slightly decreased in cells at 37?,while replication of recombinant viruses SDLY107(1-3CD)was decreased significantly at 39.5?.Cell injury induced by SDLY107(1-3CD)was more serious than that of SDLY1,but less serious than SDLY107 at 37?.However,cell injury induced by SDLY107(1-3CD)was no difference observed with SDLY1,and less serious significantly with SDLY107.Results in vitro indicated that 3D protein play important roles in replication and temperature sensitivity of EV71.4.Replication and virulence of EV71 recombinant viruses SDLY107(1-3C)had no difference with that of SDLY107 in vivo.The ICR mice injected with recombinant viruses SDLY107(1-3CD)developed less active than mice injected with SDLY107.Weight of ICR mice of group SDLY107(1-3CD)were higher than that of group SDLY107.Immunohistochemical findings suggested virus distributed in hindlimb muscle of ICR mice,and replication of group SDLY107(1-3CD)was less than that of group SDLY107.Furthermore,Pathological changes of group SDLY107(1-3CD)were less serious than that of group SDLY107.Results in vivo indicated that virulence of recombinant viruses SDLY107(1-3CD)decreased,while virulence of recombinant viruses SDLY 107(1-3C)had no difference with SDLY107.Conclusion1.Virulence of recombinant viruses SDLY107(1-3CD)decreased,while virulence of recombinant viruses SDLY107(1-3C)had no difference with SDLY107.This result indicated that 3CD protein might be responsible for virulence of EV71.2.Amino acid in 3CD protein of EV71 might act together or independently,affecting virulence of EV71,including replication and temperature susceptibility.