Contribution Of Untranslated Regions On The Virulence Of Enterovirus A71

Hand,foot,and mouth disease(HFMD)is known for a viral infectious disease,which is caused by varieties of enteroviruses.It mainly causes a concentrated infection of young children aged 5 and younger.Its typically clinical symptoms are fever,maculopapμlar rash and herpes in the hands,feet,buttocks and hips.However,meningitis,acute flaccid paralysis,neurogenic pulmonary edema and other neurological complications can be found in some severe cases,and even death.At present,enterovirus A71(EV-A71)is the major pathogens of severe hand,foot,and mouth diseases,but its pathogenesis is still unclear.According to the previous studies by our group,we speculated that untranslated regions of EV-A71 may play a role on the virulence.And the aim of this study is to determine whether untranslated regions are associated with the pathogenesis and virulence of EV-A71.Based on the reverse genetics technology,we rescued two chimeric viruses SDLY107(1-3’UTR)and SDLY107(1-5’UTR)by replacing 3’UTR/5’UTR gene fragments of an infectious cDNA clone.The virulence differences between two recombinant viruses and parental strains were studied at both cellular and animal levels to elucidate the effects of untranslated regions in EV-A71Objective:.1.To sequence and analyze the EV-A71 clinical isolates,and look for some specific mutations and clues of genetic evolution during viral transmission.2.To construct and rescue recombinant viruses by replacing UTRs of SDLY1 on the full-length infectious cDNA clone of SDLY107,and to explore the replication ability,cell damage and infectivity between the two recombinant viruses and the parental strains in vitro and in vivo.Methods:1.Extract viral RNA from nine clinical isolates of EV-A71,and the nucleotide sequences of 3TUTR was analyzed by DNAStar,BoiEdit,MEGA5.2,and Mfold.2.The recombinant fragment 3CD(107)-3’UTR(1)was obtained by overlapping PCR technology,and then substituted into the full-length plasmid of pMD19-T-107.The virus-infected RNA was transfected into Vero cells and harvested.3.Two recombinant viruses were identified by cell cytopathic effect,sequencing,and indirect immunoinfluscent assays.Then the virus titers of 50%cell culture infectious dose were measured.4.The differences of replication,cell injury rate and survival rate,and plaque forming units of recombinant viruses and parental viruses were studied in RD cells,U87 cells and SH-SY5Y cells at 37℃/39.5℃.5.The clinical manifestations of 1-day-old ICR mice were observed daily after infection with two recombinant viruses and parental viruses,respectively.The viral replication,distribution and pathological damages of brain,lung,intestine and muscle tissue were detected.Results:1.The nine isolated strains were classified into sub-genotype C4a of EV-A71,and nucleotide homology of 3’UTR was 94%~100%.The severe strain SDLY107 was different from other mild strains at 14th position,which may be related to viral pathogenicity.2.Infectious recombinant viruses SDLY107(1-3’UTR)and SDLY107(1-5’UTR)were successfully constructed after sequencing identification.Both recombinant viruses showed typical cytopathic effect after transfection,which was stable and infectious.3.The replication ability was significantly reduced when recombinant viruses SDLY107(1-3’UTR)and SDLY107(1-5’UTR)infected with SH-SY5Y cells,compared with the severe strain SDLY107.And there was no difference in replication curves between two recombinant viruses and parental strain SDLY 107 in RD cells and U87 cells.4.The cell damages of two recombinant viruses to the three cells were weaker than that of the severe strain SDLY107,especially in SH-SY5Y cells and U87 cells.5.The results of animal experiments showed that ICR mice infected with the two recombinant viruses exhibited milder symptoms than that of the severe strain SDLY107.The viral loads of SDLY107(1-3’UTR)and SDLY107(1-5,UTR)in muscles were reduced and pathological damages in brain,lung,intestine and muscles were lighter than that of SDLY107.Conclusion:1.Recombinant viruses SDLY107(1-3’UTR)and SDLY107(1-5’UTR)were successfully constructed.The replication ability and cell damage of recombinant viruses were obviously different from those of the severe strain SDLY107 in nerve cells,indicating that untranslated regions are associated with neurovirulence.2.EV-A71 infection in ICR mice is mainly characterized by muscle tropism,and UTRs are one of the factors affecting viral pathogenicity.3.The 22 positions(88nt,123nt,143nt,154nt,187nt,241nt,243nt,253nt,291nt,438nt,440nt,571nt,579nt,602nt,658nt,664nt,690nt,696nt,7328nt,7335nt,7367nt,7395nt)might be potential virulent positions of EV-A71.