| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 102 === Entervirus 71 is a major pathogen causing hand-foot-and-mouth disease or herpangina, but occasionally causes central nervous system demages. From 1998 to 2013, EV71 caused outbreaks every 3-5 years in Taiwan because of antigenic changes, but in both 2008 and 2012 years were caused by genotype B5. To examine why the same genotype EV71 could cause two large outbreaks, we examined the isolates from National Cheng Kung University Hospital to compare the full-length sequences. There were 16 amino substitutions in the non-structural region between isolates from 2008 and 2012. We compared two B5 viruses of each from 2008 and 2012, and found 2012 EV71 had higher growth kinetics and 2008 were temperature resistant. For further investigation, we constructed 2008 and 2012 3D reverse genetic (rg) virus by using N7008TW99 backbone and six single mutation viruses in 3D region. 2012 3D rg viruses had higher replication rate than 2008 3D rg viruses which was similar to native viruses. We found r3D654TW12 and r3D1745TW08 had higher fidelity by guanidine resistance assay. In sequence comparison, we found sites 383 and 396 in 3D region might play important role for EV71 replication. Furthermore, six single mutant viruses showed higher replication rate than rM202TW12 backbone virus, and low fidelity. In fitness assay, we found no difference between 2008 and 2012 substitutions. These findings suggested that the position at 383 and 396 in 3D region may be important for viral polymerase activity and replication. The results will help to understand the mechanism of EV71 replication and virulence.
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