| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 94 === Enterovirus 71 (EV71) epidemic resulted in 78 deaths in 1998 in Taiwan, with an average of 40 fatalities each year, but the molecular basis of EV71 pathogenicity remains poorly understood. Studies in poliovirus have suggested mutations in 3D polymerase region resulted in changing growth rate, viral RNA accumulation, temperature susceptibility, and attenuation in mice. Therefore, we hypothesized that 3D region may be one of the major factor altering biological characteristics and virulence of EV71. The aims of this study are to examine the effect of mutations on biological properties of 3D by site-direct mutagenesis using EV71 replicon and infectious clone systems. Two strategies were used to examine whether mutations in 3D region have the potential to affect 3D polymerase function. First, sequences of clinical isolates from 1986 and those from or after 1998 outbreaks were compared. Amino acid sequence of 3D of isolates from or after 1998 epidemic showed T251V or T251I mutation when compared with 1986 isolates. Secondly, those well-defined mutations with measurable phenotypic defects in poliovirus were examined in EV71 3D region. It was known that aspartic acid (D) in position 328, located in YGDD motif, is important for 3D polymerase activities of poliovirus. In this study, two mutations, D328H and I251T were introduced into EV71 replicon and infectious clone. The luciferase activity and virus replication rate of D328H are dramatically decreased when compared with wild type. However, this D328H mutant infectious clone was found to have a reversion from 3D-H-328 to 3D-D-328 (wild-type) at day 8 post transfection. In the I251T mutant, luciferase activities and viral replication rate showed no significant difference at 35℃ when compared with wild type, but I251T mutant showed lower luciferase activities and viral replication rate than wild type when they were incubated at 39.5℃. The results suggested that D328H mutant 3D polymerase can support a low level replication to generate a reversion, and thronine at position 251 results in strong temperature sensitivity may contribute to attenuation in virulence of clinical isolates.
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