The Study of Enterovirus 71-Induced Cell Death ; The Genetic Recombination of Enterovirus 71

• Main Authors: Shih-Chen Huang, 黃士真
• Other Authors: Wang, Jen-Ren
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/26955373206919423394

碩士 === 國立成功大學 === 分子醫學研究所 === 93 === 　Enterovirus 71 (EV71), a non-enveloped, positive strand RNA virus belongs to Picornaviridae family. EV71 is a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) in young children, and can cause severe brainstem encephalitis (BE) and pulmonary edema (PE) with high fatality rates. Cell death plays a central role in modulation of the inflammatory response during viral infection. In addition, activated T lymphocytes can release cytokines which result in apoptotic signaling pathways such as the Fas/ Fas ligand (FasL), TNFR/TNF-αlead to of cell death. The first part of study was designed to investigate of the cell death induced by the release FasL or TNF-αfrom the peripheral blood polymorphonuclear cells (PBMC) upon EV71 infection. The results showed that the concentrations of sFasL and TNF-α increased continouslly during 72 and 96 hours postinfection in EV71-infected PBMC by ELISA. However, the concentration of sFasL is not enough to cause cell death when compared with concentrations of TNF-α. In addition, adding neutralization antibody against TNF-α showed the reduction of cell death. Furthermore, inhibition of TNF-αproductionwas observed when inhibitor of NF-κB was pretreated with PBMC. The showing indicated that EV71-induced TNF-α production via NF-κB pathway which may resulted in cell death and tissue damage. 　In the second part, genetic recombination of EV71 was examined. genetic recombination is a common feature among positive-strand RNA viruses. Analysis of non-polio enteroviruses (NPEV) prototype strains has suggested that interserotypic recombination is a frequent event during natural transmission and that it may play a significant role in enterovirus evolution and virulence. To examine the role of genetic recombination in the evolution of the EV71, the partial sequences of EV71, including the 5'-UTR, VP4VP2, VP1, 2B and 3D regions, of EV71 were examined. Thirty EV71 clinical isolates before and after 1998 epidemic were compared with the homologous sequences from all other enterovirus by phylogenetic analysis using PHYLIP Neighbor-joining method. 　Four isolates (N0003-TW-05, S0584-TW-04, N3340-TW-02 and 236-TW-86) among 30 clinical isolates were identified to have the evidence of genetic mutation or recombination. Among VP4VP2, VP1, 2B and 3D sequences of two isolates from 1986 (236-TW-86 and 252-TW-86), they all belonged to genotype B. However, the 5'-UTR region of isolates 236-TW-86 belong to EV71 genotype C, indicating genetic recombination between genotype B and C in 5'-UTR region. In addition, three isolates from 2002 (N3340-TW-02), 2004 (S0584-TW-04) and 2005 (N0003-TW-05), respectively, were identified to have genetic recombination between 2B and 3D region. Sequence of 2B fragment of these isolates belonged to genotype B. However, the sequence of 5'-UTR, VP4VP2, VP1 and 3D belonged to genotype C. Using SimPlot program to check full-length sequence, the localization of recombination was identified between 3000~3500 and 5500~6000 in N3340-TW-02 isolate. 　Moreover, the virological properties of recombinated EV71 was examined, including plaque assay, one-step growth curve and temperature-resistant assay. In 39.5℃ temperature-resistant assay, the data showed that they all belong to temperature-resistant. Furthermore, N3340-TW-02 showed faster growth than non-recombinated EV71. Taken together, these results indicated that genetic recombination phenomenon may have alter their virological and virulence properties.