| BACKGROUND:Hand, foot and mouth disease(HFMD) is a common childhood acute infection,which is caused by Human enterovirus viruses, HFMD is characterized by fever, rash on the hands and feet, and mouth ulcers, occasionally accompanied by encephalitis, myocarditis, pulmonary edema, acute flaccid paralysis and other serious complications, and very few cases can lead to death. It is most commonly associated with the invasionof human enterovirus71(HEV71) and Coxsackievirus A type16. Since the first reported of HEV71in Shanghai in1981, there have been sporadic outbreaks in china. The first outbreak of HFMD was reported in Inner Mongolia Autonomous Region in2007. The prevalence of HFMD has increased greatly since2008,Up to2010, there were18944cases were reported in all, which contained247severe cases and13dead cases.But so far, the pathogensis of HFMD is uncertain, the safety and effective vaccine and drugs used for prevention and control HFMD has not been developed.OBJECTIVE:In order to prevent and control HFMD more effectively in Inner Mongolia, the characteristics of epidemiology, molecular biology of major pathogen of HFMD and the pathologic changes in a serious case caused by HEV71were studied in this thesis.METHODS:(1)Descriptive epidemiology method was used to analyze the epidemiology characteristics of HFMD patients in2008-2011in the Inner Mongolia, and the statistical software and geographic information system.(2)The clinical specimens including stools and throat swabs were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia, which were identified by using the real-time PCR method and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method (detecting EV, HEV71and CVA16in a single tube), and VP4and VP1coding region amplification and sequencing was performed with the viral isolates that were identified as non-HEV71, non-CVA16HEVs. The VP1coding regions of representative HEV71isolates that selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0software were used to analyze evolutionary relationship between representative HEV71strains and repredentative strains of each genotype/subgenotype. The full-lenth genome of12representative HEV71isolates that selected selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative HEV71strains and repredentative strains of A genotype.(3)The clinical specimens including stools, throat swabs and vesicle fluids were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method detecting CVA16; The VP1coding regions of represented CVA16isolates that selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly were amplified and sequenced. Finally the phylogenetic tree was constructed among the VP1coding regions of the different genotypes and subgenotypes of CVA16strains, and MAGE5.0software were used to analyze evolutionary relationship; The full-lenth genome of6representative CVA16isolates that selected selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative CVA16strains and repredentative strains of A genotype.(4) One serious dead case caused by HEV71was collected in Henan province in2010. The major organs were stored through formalin-fixation and liquid nitrogen respectively. An immunohistochemical techniques and histological section examination were performed to analyse the pathological features and HEV71antigen distribution.RESULTS:(1) The incidence of HFMD was sigenificant difference in difference years, months and cities during2008to2011in Inner Mongolia(P<0.05); The HFMD cases were reported in every month and the peak of incidence were abserved during June to July; the cases of HFMD were mainly scattered children and kindergarten children of10years old, especially children of5years old or younger; the male incidence was higher than female, the difference was significant(P<0.05); city incidence is higher than the incidence of towns, the difference was significant(P<0.05); CVA16and HEV71were the major pathogen of HFMD in Inner Mongolia, account for98.28%.(2) HEV71and CVA16were the main pathogens of HFMD in Inner Mongolia during2010-2011,10HEVs isolation was isolated from the case of HFMD and most severe cases were caused by HEV71. The HEV71strains circulated in Inner Mongolia were all belong to C4a evolution branch within C4subgenotype. Phylogenetic analysis revealed that Inner Mongolia HEV71strains during2010～2011located in different lineages, and has larger nucleotide identity with2008Beijing HEV71strains than2007Inner Mongolia HEV71strains. This indicated that Inner Mongolia HEV71strains had not evolved independently, but co-evolved with the HEV71strains in other provinces in mainland China; The Inner Mongolia HEV71strains have the same ancestor to the Taiwan stains; Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region.(3) CVA16strains isolated from different clinical symptoms of HFMD cases in the Inner Mongolia Autonomous Region during2010-2011belong to Bla and B1b evolution branch of B1genotype and the nucleotide acid of represented CVA16strains in Inner Mongolia were closed to CVA16strains isolated from mainland China since1998. This indicated that two evolutionary branch of CVA16in the Inner Mongolia Autonomous Region co-evolve and co-prevailed; The Inner Mongolia CVA16strains belong to the B1b evolution branch of B1genotype have the same ancestor to the Beijing stains, and has the Inner Mongolia CVA16strains belong to the B1a evolution branch of B1genotype have the same ancestor to the Thailand stains, Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71 strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region, and there was a little difference in the nucleotide acid between the Inner Mongolia CVA16strains and the prototype HEV71stain, this revealed recombination between the Inner Mongolia CVA16strains and prototype HEV71stains.(4) The dead case causing by HEV71was studied systematically. At necropsyt the changes mainly appeared in nervous system and respiratory system. Respiratory system showed congestion and edema of lungs,and the endotracheal white viscous liquid The nervous system showed brain swollen, subarachnoid hyperemia and hemorrhage. Histopathologically, the lesions were characterized by non-suppurative encephalomyelitis and viral pneumonia. The former included edema,lymphotic cuffing earound blood vessels, satellite phenomenon and neurotropic phenomenon. The later included pulmonary edeama, interstitial wideness of alveolar wall,hyaline membrane forming in alveolar cavity, a lot of macrophages, lymphocytes,pink serous exudation, and desquamated alveolar type II cells in alveolar cavity. Immunohistochemicaly,HEV71antigen signal was detected not only in nervous system,but also in lung, bronchus,stamoch, liver,duodenumjejunum, vermiform appendix,thymus, pancreas and kidney in this study.It is inferred that the HEV71is a pantropic virus; spread of the virus in the body was complicated through proliferation whithin cells(lnclede vascular enduthelium)and blood circulation;the main invasive organs were brain,spinal cord and lungs;the direct cause for death was disfunction of nervous system and respiratory failure.CONCLUSIONS:(1) Clarified the epidemiology of HFMD in Inner Mongolia during2008～2011, it was that the time of onset, susceptible populations and geographical distribution were regularity.(2) The HFMD in Inner Mongolia pathogen spectrum was mainly HEV71, CVA16and other enterovirus.(3) Revealed the major biological charateristics(including molecular epidemiology, genetic characteristics and evolutionary relationships) of HEV71and CVA16.(4)Besides nervous system,HEV71antigen signal was detected in lung, bronchus,stamoch, liver,duodenum,jejunum, vermiform appendix,thymus, pancreas and kidney by immunohistochemical staining. |
