Genomic Sequence Analysis Of Hand,Foot And Mouth Disease Virus And Preliminary Study On Endoplasmic Reticulum Stress Induced By Enterovirus 71

Enterovirus 71 is a highly pathogenic enterovirus,which mainly causes hand,foot and mouth disease(Hand,foot and mouth disease,HFMD),an epidemic in many parts of the world.The main symptoms of HFMD include hand,foot and mouth disease,buttocks and other parts of the skin rash or herpes,with death-leading aseptic meningitis,encephalitis,acute flaccid paralysis and other serious neurological complications,respiratory infections and myocarditis,etc.found in a small number of children.In previous studies,we completed the whole genome sequencing of the GDV126 virus in Hubei province in 2010.Sequence analysis revealed that it is a CVA16 strain.During the expansion of the virus,we found that it cannot infect RD cells,which are commonly used for viral amplification.This indicates that the virus may be different from other enteroviruses due to mutations.In this thesis,the phylogenetic tree was constructed using the MEGA software,based on the 5'-UTR,P1,P2,P3 regions of GDV126.Similarity plot and bootscan analyses were performed to identify potential recombination sites.The results are summarized as follows:(1)Phylogenetic analysis showed that GDV126 was clustered with EV71 genotype C4 strains for the 5'-UTR region,while the P1 region of GDV126 was independent of EV71 and CVA16.Intriguingly,the P2 and P3 regions of GDV126 virus had a high bootstrap with Coxsackie virus A16 strain XZ10-D-1.Such incongruent phylogenetic relationships observed among different regions of the viral genome suggest that recombination events may have taken place in GDV 126.(2)The similarity plot analysis showed that GDV 126 had a high sequence similarity(?94%)to EV71 genotype C before position 2240,but a higher similarity(?96%)to CAV16 after position 2240.The bootscan analysis from 5' end of the genome to position 2160 showed a high bootstrap support for clustering between EV71 genotype C and GDV126,whereas that from position 2280 to the 3'end of the genome showed a high bootstrap support for clustering between CAV16 and GDV 126.These findings indicate that recombination events are likely to have taken place between the nucleotide 2160 and 2280,corresponding to the VP3 region that is linked to the virulence.In recent years,there are many outbreaks in many regions of China.In the past four decades,EV71 virus gene has evolved.Thus,the investigation of its genetic variations has important significance for the early diagnosis,typing and understanding of the pathogenesis of this epidemic.The endoplasmic reticulum(ER)is the main post-translational site for protein folding.A large number of viral proteins are synthesized in the process of viral infection.The increased load of unfolded or misfolded proteins can activate the ER stress response,and the subsequent signaling pathways regulate the cell survival or cell death.The results are summarized as follows:(1)Analysis by Western blot and real-time quantitative PCR showed that EV71 virus infection could activate the IRE1?-XBP1 and PERK pathways of ER stress.Preliminary results indicate that the 3D protein of EV71 may affect the phosphorylation state of IRE1?.(2)In cells infected by EV71 virus,decreased IRE1? protein levels were observed,which did not result from changes in its mRNA abundance.(3)Analyses by TCID50,Western blot,real-time quantitative PCR showed that inhibition of IRE1? phosphorylation appeared to enhance viral replication,while inhibition of its endonuclease activity might reduce virus replication.This implies a possible role of IRE1? phosphorylation in regulating viral replication.The study of ER stress signaling pathways in the regulation of the antiviral inflammatory response will help us gain further insights into the molecular pathogenesis of EV71-related HFMD.