MEASLES CIS-ACTING DETERMINANTS OF VIRULENCE

麻疹毒力的顺式作用决定因素

• 批准号: 2070862
• 负责人: Stephen A. Udem
• 金额: $ 2.82万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1994-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2070862
• 关键词: autoradiography; fusion gene; genetic regulatory element; genetic strain; genetic transcription; luciferin monooxygenase; measles; measles vaccine; measles virus; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; transfection; transfection /expression vector; virulence; virus replication

Epidemic measles recently resurfaced in the U.S., raising public health concerns about current vaccine strategy and design and doubts that eradication of this serious acute viral infection can be attained by the turn of the century. While measles resurgence largely can be ascribed to failure to achieve age-appropriate mass immunization, additional features of measles epidemics throughout the world point to deficiencies in the current vaccine's efficacy. Clearly, a new generation of measles vaccines that induce abundant, long-lived immunity even when administered to infants 6-9 months of age is internationally needed. Intelligent vaccine design, however, must rest on a far better understanding of measles virus (MV) and in particular, on the as yet unknown identity of the virally encoded determinants of virulence as well as of those genomic changes that lead to attenuation. This proposal seeks to address the hypothesis that important virulence/attenuation determinants reside in viral genomic non- protein coding regulatory regions: the 3' and 5' cis-acting putative promotor and/or regulatory sequence elements directing genomic transcription, genome and antigenome encapsidation, and replication; and the short regions encompassing internal intergenic boundaries specifying transcription termination and reinitiation. Thus, specific nucleotide changes in these regions may affect virulence by modulating transcriptional and/or replicative efficiency thereby determining the abundance of cytopathic viral gene products and/or virion progeny. To address this hypothesis, the nucleotide sequences of the non-protein coding regulatory regions of various epidemiologically distinct wild-type MV isolates as well as vaccine strains will be determined. Unique nucleotide changes found in the genomic regulatory regions of these MV strains will be tentatively assigned as virulence or attentuation determinants. Validity of that tentative assignment will be appraised b determining the influence of these nucleotides on transcription and replication when incorporated into a newly developed measles 'minireplicon', a chimeric genome sense RNA consisting of the MV 3' and 5' nonprotein coding sequences flanking an antisense firefly luciferase coding sequence from which luciferase mRNA is transcribed and new MV:luciferase minigenomes replicated when introduced into helper-cells. Evaluating the role of distinctive MV strain nucleotide changes in intercistronic regulatory domains will be approached by a similar strategy upon completion of construction of a bicistronic artificial MV: reporter gene minigenome now underway. Finally, the means must be developed by which such virulence/attenuation determinants can be introduced into a predefined measles sequence from which new genetically altered virion progeny can be propagated and tested for their efficacy as a live attenuated vaccine. To that end, continued effort will be directed towards refining the vector and expression system by which genetically modified infectious MV strains can be produced from full-length MV cDNA.

流行病最近在美国浮出水面，提高了公共卫生 担心当前的疫苗策略和设计和怀疑 消除这种严重的急性病毒感染可以通过 世纪之交。尽管麻疹的复兴很大程度上可以归因于 无法实现适合年龄的质量免疫，其他功能 全世界的麻疹流行病表明缺陷 当前疫苗的功效。显然，新一代麻疹疫苗 即使对 每6-9个月大的婴儿在国际上是需要的。智能疫苗 但是，设计必须基于对麻疹病毒的更好理解 （MV），尤其是在病毒的尚未知道的身份 毒力的编码决定因素以及那些基因组变化 导致衰减。该提议旨在解决以下假设 重要的毒力/衰减决定因素存在于病毒基因组非 - 蛋白质编码调节区域：3'和5'顺式作用推定 引导基因组的启动子和/或调节序列元件 转录，基因组和抗原组封装和复制；和 包括内部基因间边界的短区域 转录终止和重新激活。因此，特定的核苷酸 这些区域的变化可能会通过调节来影响毒力 转录和/或复制效率，从而确定 细胞病毒基因产物和/或病毒体后代的丰度。 为了解决这一假设，非蛋白质的核苷酸序列 各种流行病学上不同野生型的编码调节区域 将确定MV分离株以及疫苗菌株。独特的 这些MV的基因组调节区域中发现的核苷酸变化 菌株将暂时分配为毒力或注意力 决定因素。暂定任务的有效性将被评估b 确定这些核苷酸对转录和 重复纳入新开发的麻疹时 “ Minireplicon”，一种由MV 3'和5'组成的嵌合基因组感官RNA 非蛋白质编码序列侧面是反义萤火虫荧光素酶 从中转录荧光素酶mRNA并新的编码顺序 MV：引入辅助细胞时复制的荧光素酶小素。 评估独特的MV菌株核苷酸变化的作用 类似策略将通过类似策略来接触我的间际监管域 建造双科斯特人人工MV时：记者 现在正在进行的基因微型元组。最后，手段必须由 哪种毒力/衰减决定因素可以引入 预定义的麻疹序列，从中改变了遗传学的病毒体 后代可以传播并测试其作为活的功效 疫苗减弱。为此，将继续努力 完善基因修饰的矢量和表达系统 可以通过全长MV cDNA产生感染性MV菌株。