应用兔戊型肝炎病毒感染性cDNA克隆研究其复制及致病特点

批准号:
81601769
项目类别:
青年科学基金项目
资助金额:
17.0 万元
负责人:
刘鹏
依托单位:
学科分类:
H2103.肝炎病毒与感染
结题年份:
2019
批准年份:
2016
项目状态:
已结题
项目参与者:
韩扬、张栋、张国杰、岳永松、裴雨晴
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中文摘要
兔HEV是近年来发现的新型人畜共患病原体,在我国广泛分布,然而我们对其复制和致病特点还知之甚少。前期研究发现所有兔HEV的Macro Domian均有31个氨基酸(31aa)的插入,而其他类型HEV没有,表明这31aa对于兔HEV至关重要。为探明这31aa与兔HEV复制和致病性的关系,本研究拟应用申请人博士阶段构建的兔HEV感染性cDNA克隆,首先特异性缺失、替换及定点突变兔HEV特有的31aa,继而转染人源和兔源细胞系,最后通过流式细胞术、免疫荧光染色和荧光素酶报告基因等手段阐明31aa在兔HEV复制周期中的重要性;通过肝内注射RNA转录本感染SPF兔后,检测动物的ALT、病毒血症和粪便排毒持续时间和HEV载量、抗体转阳时间以及肝脏病理改变等指标来探究缺失31aa对病毒致病性的影响。本研究成果将有助于加深对兔HEV的复制和致病机制的了解,并有望为发展新的抗HEV策略提供理论和实验依据。
英文摘要
Recently, rabbit HEV strains have been isolated and shown to be prevalent throughout much of mainland China. However, much about the replication mechanisms and pathogenesis of the virus is still poorly understood. Previous studies revealed that a 31aa insertion in the macro domain of the ORF1 protein was found among all the rabbit HEV strains, but not any other strains from genotypes 1-4, which suggested that the 31aa insert is crucial for rabbit HEV. In order to determine the role of the 31aa insert in the viral life cycle, mutagenesis studies will be performed by deletion, substitution or site-mutation of the 31aa using an infectious cDNA clone of rabbit HEV generated during my PhD study. The RNA transcripts of the wild-type virus or those mutants will be transfected into human Huh7 and rabbit RK13 cells and their replication levels will be compared by flow-cytometry, immunostaining and Gaussia luciferase system, which will demonstrate the importance of the insert in viral replication. Those RNA transcripts will also be injected directly into the liver of SPF rabbits and viral infectivity and pathogenesis will be monitored by measuring ALT level, duration of viremia and fecal virus shedding, HEV viral load, seroconverted time-point and liver pathology of infected animals. Comparison of indicators above will ascertain the impact of 31aa deletion on viral pathogenesis. The results of this study will contribute to the understanding of mechanisms of rabbit HEV replication and pathogenesis, and provide theoretical and experimental evidence for developing strategies of HEV prevention.
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