Development of Japanese Encephalitis Virus Live Attenuated Vaccine and Study of the Roles in Glycoproteins E or NS1 of JEV in Virus-induced ER Stress
Autor: | Ching-Yu Liang, 梁靖瑜 |
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Rok vydání: | 2006 |
Druh dokumentu: | 學位論文 ; thesis |
Popis: | 94 In recent years, mosquito-borne infections are reemerging or emerging worldwide at an alarming rate. Thus, their vaccine development remains a top priority on the public health agenda. The current licensed live attenuated vaccines against flaviviral infection include yellow fever virus YF-17D and Japanese encephalitis virus (JEV) 14-14-2. Characteristically, human are the dead-end host for JEV infection, but not YFV, because of insufficient viremia. Therefore, we use JEV genomic RNA as a backbone to develop chimeric live attenuated vaccine against other flaviviral infection. Hopefully, the chimeric viruses may be able to trigger a protective immunity but incapable of transmitting from human back to mosquitoes. We have replaced the structural or non-structural proteins from other flavivirus into JEV genomic RNA, and then evaluate the vaccine capability of these chimeric viruses. To prevent the given mutant viruses revert to wild-type strain, we combine at least two attenuated markers into one backbone of JEV infectious clone by site-directed mutagenesis. So far, we have not generated any such chimeric viruses, and the construction process is currently underway. ER is the primary site of flaviviral glycoprotein synthesis, viral genomic RNA replication, and virus particle maturation. ER stress-mediated signaling has been implicated in the JEV-induced apoptosis. We in this study investigate how the ER stress could be induced by mutant E or NS1 proteins. ER stress was monitored by analysis XBP1 splicing signal using RT-PCR and Luciferase reporter system. We found mutant viruses P0004 (E, S331R) and P0006 (E, Q52K) could obviously induce less ER stress compared with other mutants and the wild-type RP9/X. Additionally, mutant viruses P0011 (NS1, P250L) and P0016 (NS1, T132A) induce stronger ER stress than wild-type RP9/X. To exclude other viral proteins affect ER stress, we have individually expressed E or NS1 protein to clarify the correlation between attenuate makers and ER stress. We found the E mutant protein induce stronger ER stress than the wild-type E, whereas NS1 mutants of glycosylation (P0016) or dimerisation (P0011) induce stronger ER stress than the wild-type. The result suggests that JEV glycoproteins E and NS1 have important role of virus-induced ER stress. Virus may regulate UPR by E or NS1 protein to influence cell repair or apoptosis, and further to influence replication and assembly of virus itself in cell. |
Databáze: | Networked Digital Library of Theses & Dissertations |
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