TOGAVIRUS TROPISM FOR BONES, JOINTS, AND CNS

人类感染托加病毒的特点是急性和持续性关节炎/关节痛以及病毒性脑炎。saa . ar86是毒虫科的一种甲病毒，成年小鼠感染saa . ar86为研究这两种人类疾病病理的分子基础提供了一个优秀而独特的模型系统。这些模型的引人注目的特点包括：a) S.A.AR86和相关病毒引起人类急性和持续性关节痛。b)我们对saa . ar86及其几个近缘种进行了基因组测序，构建了saa . ar86真品感染性cDNA克隆，并成功利用基于saa . ar86的表达系统进行了体外和体内实验。c)初步数据显示，在小鼠骨和关节组织外周接种并在这些组织中持续至少3个月后，急性saa . ar86趋向性。d)与其他sindbis - α病毒组成员不同，S.A.AR86在所有年龄的小鼠中都具有神经毒性，并且这种表型映射到负责RNA合成的病毒非结构基因。我们提出以下具体目标：1)确定骨/关节组织中S.A.AR86复制的细胞靶点，表征S.A.AR86在这些组织中的持久性，并评估其他与关节炎/关节痛相关的托加病毒，包括风疹和罗斯河病毒，在骨/关节组织中的复制和/或持久性。2)通过将S.A.AR86基因组中的密码子改变为非神经毒性病毒中的密码子，并筛选神经毒性丧失，将评估非结构基因中9个假定的神经毒性遗传决定因素。此外，候选的S.A.AR86密码子将被引入到非神经毒性病毒基因组中并筛选获得的毒力。3)突变调节成年小鼠神经毒性的机制将通过Aim 2中鉴定的非结构基因突变以及已经鉴定、克隆和部分表征的nsP1 538突变来检验。

Togavirus infection of humans is characterized by acute and persistent arthritis/arthralgia, as well as viral encephalitis. Infection of adult mice with S.A.AR86, an alphavirus in the family Togaviridae, provides an excellent and unique model system to examine the molecular basis for both of these human disease pathologies. Attractive features of these models include: a) S.A.AR86 and related viruses cause acute and persistent arthralgia in humans. b) We have sequenced the genomes of S.A.AR86 and several of its closest relatives, constructed an authentic S.A.AR86 infectious cDNA clone, and successfully utilized expression systems based on S.A.AR86 for in vitro and in vivo experiments. c) Preliminary data demonstrated acute S.A.AR86 tropism for mouse bone and joint tissue following peripheral inoculation and persistence in these tissues for at least 3 months. d) Unlike other members of the Sindbis-group of alphaviruses, S.A.AR86 is neurovirulent in mice of all ages, and this phenotype maps to the viral nonstructural genes responsible for RNA synthesis. We propose the following Specific Aims. 1) Identify cellular targets of S.A.AR86 replication within bone/joint tissue, characterize S.A.AR86 persistence within these tissues, and evaluate other arthritis/arthralgia associated Togaviruses, including rubella and Ross River virus, for replication and/or persistence in bone/joint tissue. 2) Nine putative genetic determinants of neurovirulence within the nonstructural genes will be evaluated by changing these codons within the S.A.AR86 genome to the codons found in non-neurovirulent viruses and screening for loss of neurovirulence. In addition, the candidate S.A.AR86 codons will be introduced into non-neurovirulent virus genomes and screened for gain of virulence. 3) The mechanism(s) by which mutations modulate neurovirulence in adult mice will be examined using the nonstructural gene mutants identified in Aim 2, as well as a mutation at nsP1 538, which has already been identified, cloned and partially characterized.