Isolation And Identification And Preliminary Study On Pathogenic Mechanism Of EV71

Enterovirus71(EV71), a member of the genus Enlerovirus of Picornaviridae family, causes major outbreaks of hand, foot, and mouth disease(HFMD), most frequently affecting children. The patients hospitalized with mild EV71infection present clinical symptoms such as slight fever, vesicular lesions. Critically ill patients present severe central nervous system (CNS) symptoms such as aseptic meningitis, brainstem encephalitis, poliomyelitis-like paralysis, et al. It has a high fatality rate. Up to now, the molecular pathogenesis of KV71infection is still unstated. The generation of the different clinical symptoms may be due to the differences of nerve virulence or the different individual susceptibility.HV71strains were collected from the HFMD patients’stool specimens and isolated in RD cell. Analysis of the complete genome of HV71could find some differences in sequences. Aiming to find the KV71pathogenic mechanism in the nervous system lesions, our study based on the regions of5’UTR,VP2and3D. The result could provide the important foundation for clarifying the virus pathogenic mechanism.Objective1. To isolate EV71from the HFMD patients’stool specimens, which will provide a basis of sequence study.2. To understand the complete genome sequences’ characteristic of KV71strains isolated in Linyi, Shandong Province, and find differences of genome sequences in strains from the patients with different clinical symptom, which will provide the candidate loci for further study.3. To research the EV71pathogenic mechanism in the nervous system lesions based on the candidate loci of5’UTR, VP2and3D.Methods1. Enterovirus was isolated in RD cell, collected from the patients’ stool specimens in Linyi, Shandong Province in2009～2010. Using enterovirus general primers and EV71specificity primers, EV71was identified by RT-PCR.2. According to the genome sequences of EV71in GenBank, we designed the primers for amplication of SDLY11. Using DNA star and MEGA4software, we analysed the complete genome sequences of EV71.3. On the basis of genome sequences of SDLY01, SDLY11, SDLY48, SDLY96, SDLY107and SDLY153, we designed primers for5’UTR and amplified the5’ UTR of8strains by RT-PCR. We analysed the sequences of5’UTR by software.4. We designed primers for VP2,3D and amplified these regions of SDLY11and SDLY107by RT-PCR. We bulit the expression vectors VP2-pBluescriptSK(+),3D-Flag-pcDNA3.1and detected proteins’ cytopathic effection by transfection in vitro. LDH experiment tested about cell injury of VP2and3D protein. Using Annexin-V and PI, we could know the cell apoptosis caused by3D protein. MTT experiment could detect cell proliferation caused by3D protein.Results1. Among24isolates,20were EV71and4other enteroviruses in2009-2010.18strains were EV71in22strains isolated in2009(81.8%).2. The genome of SDLY11had7405nucleotides,5’UTR contained742nt,3’UTR contained84nt and ORF contained6579nt, encoding a polyprotein with2193amino acids. SDLY11belonged to C4subgenotype by sequence analysis. Two mutations (T40C,C575T) in5’UTR and one mutation (T144S) in VP2were found in strains from patients with different clinical symptoms. 3. Length of5’UTR for8strains were741-745nt, the sequences exhibited highest homology to the subtype C4of EV71. Two mutations(G265A,G703T) were found in strains from patients with different clinical symptoms in5’UTR.4. VP2protein from SDLY11and SDLY107could be expressed in Vero cells, the result of LDH showed that two groups had no difference in cell injury.5.3D protein from SDLY11and SDLY107could be expressed in RD cells, LDH experiment showed3D protein transfection group of SDLY11had more cell injury than3D protein transfection group of SDLY107. Apoptosis in RD cell had no difference. MTT experiment showed that3D protein transfection group of SDLY107had stronger cell proliferation than3D protein transfection group of SDLY11.Conclusion1. The popularity of HFMD is mainly caused by EV71in Linyi, Shandong Province. SDLY11belongs to C4subtype, which is the popular subtype of KV71in mainland China.2.4mutations (T40C,G265A,C575T, G703T) in5’UTR may be associated with nervous system symptoms caused by LV71. These will provide the candidate sites for further research.3. VP2protein of strains from patients with different clinical symptoms may not cause cell damaging. However,3D protein has great influence in cell damage and cell proliferation. These results provide an important basis for the study of HV71pathogenesis mechanism.