| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 101 === Enterovirus 71 (EV71) is a non-enveloped, positive-strand RNA virus of the Picornaviridae family. The viral genome encodes a single polyprotein precursor of 2,193 amino acids. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3 and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C and 3D) proteins. Several outbreaks were reported worldwide since it had been isolated firstly in California in 1969 known as EV71 genotype A. Studies on EV71 epidemiology showed that genotype B3-B5 and C2-C5 have caused large outbreaks in the Asia-Pacific region since 1997. During 1998, EV71 genotype C2 burst out the biggest outbreak in Taiwan, resulting in 405 cases of severe neurological sequelae and 78 deaths. In addition, in 2008 and 2012, genotype B5 has caused serious HFMD epidemics in Taiwan. In this study, we aim to find genetic variations between EV71 isolates in 1998, 2008 and 2012 by analyzing EV71 full-length sequences. First of all, we found no conserve difference among severe and mild cases in 2008 or 2012. Interestingly, there were several different amino acids in non-structure proteins between 2008 and 2012. These differences in non-structure proteins may correlate with the re-emergence of EV71 genotype B5 in 2012. In 1998, the 9 fatal and 15 non-fatal cases were used to produce full length clone and sequence. Two major nucleotide differences among fatal and non-fatal cases were identified at the position 4985 in 2C302 and position 5555 in 3C55. In fatal cases, leucine was identified more than valine at 2C302 and more valine than isoleucine was found at 3C55. In non-fatal cases, the frequency of amino acid expressing is opposite. The percentage of leucine and valine which expressed simultaneously in 2C302 and 3C55 is 56% among fatal cases and only 27% among non-fatal cases. In order to examine the functional role of 2C302 and 3C55, we introduced the 2C-L302V and 3C-V55I mutations, single or double, by site-directed mutagenesis to the EV71 full length infectious cDNA clone. Reverse genetics viruses (rg viruses) obtained were used to examine viral properties. The four rg viruses all showed temperature sensitive phenotype and induced similar effect of apoptosis. However, the rg virus 6359, which contained 2C-L302 / 3C-V55 showed faster growth than other mutant viruses on one step viral growth curve at high moi in both RD and SK-N-SH cells. In addition, the 6359 rg virus formed larger plaques compared to that of mutant rg viruses. In addition, it has been demonstrated that nitric oxide (NO) could inhibit the replication of many pathogens, including EV71. We found the 2C302 and 3C55 can affect the sensitivity of inhibition by NO. This study suggests amino acids at 2C302 and 3C55 play various roles in EV71 replication.
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