Etiology And Clinico-epidemiological Feature Of Hand, Foot And Mouth Disease Caused By Human Enterovirus71in Guangdong2009-2011and Production Of Monoclonal Antibodies To P1

Hand-foot-and-mouth disease (HFMD), characterized by fever and acute vesicular eruptions of palms, soles of the feet and mouth (herpangina), is a common exanthema in young children. It emerged as a signicant public health threat in the Asia-Pacific region with large outbreaks occurring frequently recent years and increased concern of fatal HFMD cases. HFMD is caused by members of the non-polio Enterovirus genus (family Picornoviridae) and human enterovirus71(EV71) is one of the main etiologic agent which could cause severe neurological complications. EV71is a single-stranded, positive-sense RNA virus whose genome(～7500bp) consists of one upen reading frame(ORF,～6600bp) is expressed as large polyprotein that can be divided into3regions:P1, P2and P3.The four structural proteins, VP1, VP2, VP3and VP4. are encoded within P1region. Currently, there is no vaccine available and treatment is limited to palliative care. Molecular biology and pathology are essential to a fundamental understanding of pathogenesis of HFMD for vaccination and treatment purposes. We aimed to, first, present an empirical analysis of the etiology and clinico-epidemiological data concerning the HFMD cases caused by EV71in Guangdong during2009～2011; Second, understand the kinetic profiles of the IgM antibodies and the diagnostic characteristic of the IgM-capture ELISAs for HEV71for diagnosis of HFMD in pediatric patients; Third, produce monoantibodies to polyprotein P1:VP1, VP2, VP3and VP4, the capsid proteins fpr further identifying the epitopes. The study was divided into three parts:First part Etiology and clinico-epidemiological feature of HFMD caused by human enterovirus71in Guangdong2009-2011.The present study aimed to the detection of the EV71involved in the epdemic during2009-2011in Guangdong and the characteration of the clinico-epidemiological feature of HFMD caused by human entervirus71(EV71) among the HFMD cases in Zhujiang Hospital. The main methods we used for etiology diagnosising of HFMD are detecting specific virus RNA in retacal swabs by real time RT-PCR, anti-EV71IgM in acute serum by captured ELISA, neutralization test for paired sera, semi-nested-RT-PCR and sequencing for seratyping and virus isolation and characterization.Totally1239(Male813, Female426, ratio1.91:1) were dignosised HFMD in Zhujiang Hospital in2009-2011, each year107,553,579cases were reported. The positive rate of EV were87.9%(94/107),91.2%(505/553) and88.8%(515/579) respectively and that of EV71were28.1%(30/107)、45.6%(252/553) and38.7%, suggesting that EV71was the main etiologic agent of HFMD in Guangdong in recent years. Ages of patients ranged from1month to35years, most were children, expecially aged from1to4years old (85.0%). Totally21patients were under six month ameng them eightt were caused by EV71. Eighty-one patients occured severe CNS complications with no significant difference of age distribution between HFMD with and without CNS complications. In Guangdong, there were two peaks for HFMD outbreaks each year:May and late Novenber and early December. Vesicular eruptions (90%) and fever (60%) were the most frequent manifetions in acute phase and CNS infections are the main complications followed by respiratory complications. Thirty-there (5from2009,28from2010) isolates from HFMD patients were amplified VP1fragments (～600bp) and were genotyping as C4a. The phylogenetic tree was constructed indicating the strains circulating in Guangdong were much likely from mainland China which different from Taiwans’. Analysis of similarity alignment of amino acid sequences of VP1between Guangdong2009-2010, Taiwan98and standard strain BrCr (U22521) only22th and98th anmino acids were difeerent.EV71was the main pathogen of the epedimc of HFMD in2009-2011of Guangdong. EV71is also the cause of most severe cases that more attention should be taken to prevent its infections.Second part Evaluation of human enterovirus71specific immunoglobulin M antibodies for diagnosis of HFMD.Reliable methods are needed for diagnosis of HFMD in childen. IgM-capture ELISA, with its notable advantages of convenience and low cost, provides a potentially frontline assay. We aimed to evaluate the newly developed IgM-capture ELISAs for EV71in the diagnosis of HFMD, and to measure the kinetics of IgM over the course of HEV71infections. We mapped, for the first time, the kinetics of IgM in EV71infection. EV71-IgM was detectable in some patients on day1of illness, and in100%of patients by day5and persisted for several weeks. The IgM detection rate was90.2%(138of153sera) for EV71infections during the first7days of diseases. During the first90days after onset these values was93.6%(233of249sera). Some cross-reactivity was observed between EV71-and CA16-IgM ELISAs. To further analyze the cross-reactivity of the assays, a total of122sera from66CA16,49sera from other enterovirus and105sera from75other respiratory virus infected patients were tested for both EV71-and CA16-IgM simultaneously. EV71-IgM was positive in38of122(31.1%) CA16infections,14of49(28.6%) other enteroviral infections and2of105(1.9%) for other respiratory virus infected sera. Nevertheless, the ELISAyielded the higher OD450value of main antibody than that of cross-reaction antibody, successfully identifying the enteroviral infection in96.5%(EV71) cases. When blood and rectal swabs were collected on the same day (111EV71) the data showed that the agreement between IgM-capture ELISA and real-time RT-PCR in EV71was high (Kappa value=0.729). The sensitivity for EV71-IgM was90.1%(95%confidence interval:84.1-94.4%). These result suggested capture-ELISA perform as well as RT-PCR in diagnosing HFMD and could be deployed successfully in clinical and public health laboratories.Third part Production of monoantibodies to monoantibodies to polyprotein P1of EV71.The open reading frame(ORF,-6600bp) of EV71expressed as large polyprotein that can be divided into3regions:P1, P2and P3. The four structural proteins, VP1, VP2, VP3and VP4, are encoded within P1region which are the main antigen epitopes of the virus. We aimed to produce monoantibodies to polyprotein P1: VP1, VP2, VP3and VP4for further study on molecular biology and pathology of EV71. Full length VP1to VP4genes were amplified by PCR then the products were digested by double restriction enzyme:KpnW Ⅰ and Hind Ⅲ, then ligated to the vector of pQE30a and transfected to the expression system of E coli. M15with IPTG induction. Recombinant VP1～4-His fusion proteins were harvested followed by affinity purification. The recombinant VP1～4-His fusion proteins were all in the status of inclusion body, they were involved in8M urea for denaturation. The virus particals of EV71were purified by centrifugation in sucrose gradient.Both the denaturated recombinant VP4protein and EV71particals were used as immunogen to injected mice. After the fusion cells were harvested, ELISA for EV71and recombinant VP4protein were used for screening the clones, the positive clones were further identified by EV71in RD cells by indirect immunofluorescene assay. The positive clones by both assays were then characterized by Western-Blot for both virus and recombinant VP1～4-proteins revealed by15%SDS-PAGE. At last we acquired25clones among which six were for VP1, three for VP2, two for both VP1and VP2, one for VP4and13untyped.Summarization:According to the results of three parts above, we summarize as follows:1. EV71was the main etiologic agent of HFMD in Guangdong during2009-2011,with positive rate of EV71detecting were28.1%(30/107)、45.6% (252/553) and38.7%in each year respectively.2. There were two peaks for outbreaks of HFMD each year in Guangdong:May and late Novenber and early December. No community outbreaks were reported in our study. Age ranged from1to4years children (85.0%) were the main part of HFMD patients. Patients occured severe CNS complications with no significant difference for age distribution between HFMD with and without CNS complications.3. Thirty-there isolates from HFMD patients in2009-2010were genotyped as C4a. The phylogenetic tree was constructed indicating the strains circulating in Guangdong were much likely from mainland China which different from Taiwans’4. We mapped the kinetics of IgM in EV71infection that EV71-IgM was detectable in some patients on day1of illness, and in100%of patients by day5and persisted for several weeks. EV71IgM-capture ELISA was as well as RT-PCR in diagnosing HFMD and could be deployed successfully in clinical and public health laboratories.5. Though the cross-reactivity of the assays between EV71and CA16were observed, the ELISA yielded the higher OD450value of main antibody than that of cross-reaction antibody, successfully identifying the enteroviral infection in96.5%(EV71) cases.6. Using the recombinant VP4protein and EV71particals as immunogen to injected mice,we finaly got25clones among which six were for VP1, three for VP2, two for both VP1and VP2,1for VP4and13untyped...