Study Of Mechanism,Laboratory Indicators And Pathogen Of Mild And Severe Patients With Hand,Foot And Mouth Disease

Objective1.To compare and analyze the difference of laboratory indicators between mild and severe patients with hand,foot and mouth disease whose clinical characters and laboratory indicators were investigated from a children hospital of Zhengzhou city in 2015,and to provide evidence for predication of patients' severity.2.To compare and analyze difference of the coding region between mild and severe patients' enterovirus 71 with hand,foot and mouth disease after describing the characters of clinically isolated pathogens.3.To study the relationship between oxidative stress and cytokines in RD cells infected by enterovirus 71.Methods1.General information and laboratory indicators were collected from 187 HFMD patients by questionnaire.Differences between mild and severe HFMD patients were compared by univariate and multivariate analysis.2.A total of 748 enterovirus with complete genome were collected from Genbank database,but only 230 strains had definite clinical background after literature retrieval.Then their molecular characters,amino acid site,secondary structure and domains were analyzed by bioinformatics software.3.Levels of reactive oxygen,phosphorylate and unphosphorylate NF-kappaB and IL-6,TNF-? were detected by using spectrophotometry measure,Western blot and ELISA in RD cells at 0h,12 h,24h,48 h post infection respectively.Results1.Neutrophils and lymphocyte ratio,IgM and procalcitonin changed dynamically in HFMD patients(P<0.05);Neutrophils(OR=1.198,P=0.001)and lymphocyte ratio(OR=1.194,P=0.002,leukocyte count(OR=0.916,P=0.024,serum sodium(OR=0.690,P<0.001)might affect or reflect the severity of HFMD.2.A total of 103 strains and 127 strains were collected from mild and severe HFMD patients respectively,which mainly consisted of genotype C4 and were isolated in the Asia-Pacific region.The similarity between strains among same time or regions were bigger than that among different time or regions.The homology of VP1 amino acid among EV71 strains of mild and severe patients was 96%-100% and 94%-100% respectively,while the homology of VP1 amino acid among both EV71 strains was 92%-100%.The distributions of 41 amino acid sites between mild and severe patients' strains were statistically different(P<0.05),and 26 different amino acid sites were located in P3 region which had more changes(P<0.001).Moreover,21 and 20 amino acid sites also were distributed in both secondary and non-secondary structures,but none were in functional domains.3.The levels of reactive oxygen increased statistically comparing among 24 h and 48 h groups with 0h group(P<0.05).More NF-kappaB was activated and then transported to the nucleus comparing 48 h group with 0h(P=0.0394).In contrast with 0h infection group,levels of IL-6 in groups increased statistically significantly at 12h?24h and 48 h post infection(P<0.05).Meantime,levels of TNF-? increased statistically comparing 48 h group with 0h group(P<0.05),while no significance was found comparing 12h?24h group with 0h group.Conclusions1.The immune level changed dynamically in HFMD patients,which might affect the severity outcome.EV71 genotypes were different among different time or regions;The homology was low between strains from mild and severe HFMD patients.2.Substitutions of amino acid might influence protein functions by changing primary and secondary structures,which might affect phenotype of enterovirus 71.3.Oxidative stress was induced in RD cells after infection by EV71.NF-kappaB was activated and increased levels of IL-6,TNF-?.