| Enterovirus71(EV71) is the main causative agent of hand-foot-and-mouth disease(HFMD). The infection of EV71can cause neurological symptoms [1]. The research on thevirulence and pathogenic mechanism of EV71has vital significance on the prevention andtreatment of HFMD [2,3]. We isolated EV71strains from severe HFMD patients and haslaid the foundation for EV71animal model building, pathogenesis research and vaccineresearch through the genetic analysis, and rescue virus of EV71.In this study, two strains of virus were isolated from throat swab specimen of theinfected children suffering severe neurologic symptom and were identified as EV71bynucleic acid detection and ATCC enterovirus standard serum neutralization test. Twostrains of EV71were cloned out through virus plaque test and named as EV71-961andEV71-102. By comparing these quencing results of VP1region with the existing sequencesin the Genbank, the two EV71strains were classified into EV71genotype C4. Accrodingto the sequence analysis, full lengths of both two strains of virus constructed were7407nt.The EV71-961shared a maximum genome homology of97.72%(7233/7402) withChongqing3-09-china (Genbank Number: GQ994991.1, Cq3). The EV71-102shared amaximumgenome homology of99.75%(7188/7206) with Henan1-09-China (GenbankNumber: GU196833.1, Hn1). The further genome analysis found that the homology of1-3760nt and3761-7407nt between EV71-961and Cq3and Hn1were99.87%（3756/3761） and99.97%(3556/3557), respectively. The EV71-102shared a maximumhomology on5’NTR (98.65%,730/740）with Guangdong2009（Genbank Number:JF799986.1，Gd2009）, and there were only5sites differed from Hn1in the structuralprotein. Accordingly, it can be concluded that those two strains of virus are therecombinant virus of Hn1with Cq3and Gd2009. Among them, Hn1is the velogenic strainand Cq3is the low virulent strain [4]. In order to conductin-depth researches on those twostrains, the technology of reverse genetics was applied to construct the infective clones of EV71-961and EV71-102.The constructed infective clones transfected the Vero cells to respectively obtain therescue virus of EV71-961and102. The result of virus sensitive test in different cells wasVero＞SN-SK＞RD＞Hela. The determination of virus titer（TCID50）revealed a similarityof titer between rescue virus and mother virus, both around7.0lgTCID50/ml. In thefollowing research, the apoptosis experiment and one step growth curve were inconsistency with the cytopathy observed via microscope. The virus was observed in thecytoplasm of the positive cells by the immunofluorescence test.Toxicity and immunogenicity results showed that the fatality rate of original EV71strain infected rats was higher than that of rescue viruses infected rats. Amone them, thefatality rates of EV71-102infected rats were higher than that of EV71-961infected rats.Our study showed that the virus gene recombination could lead to the change of thevirus virulence, the more genome restructured, the more virulence changed. Virus immunemice produced strong humoral immune response.The purer the virus stimulated the body,the higher the antibody concentration produced.The infection cloning constructed in this study and the rescue virus obtained layfoundation for the following research on the animal models, pathopoiesia mechanism andvaccine of EV71. |
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