TOGAVIRUS TROPISM FOR BONES, JOINTS, AND CNS

人披膜病毒感染的特征在于急性和持续性关节炎/关节痛以及病毒性脑炎。用披膜病毒科中的甲病毒S.A.AR86感染成年小鼠，提供了一个极好的和独特的模型系统来检查这两种人类疾病病理的分子基础。 这些模型的吸引人的特征包括：a）S.A.AR86和相关病毒在人类中引起急性和持续性关节痛。B）我们已经测序了S.A.AR86及其几个最接近的亲属的基因组，构建了一个真实的S.A.AR86感染性cDNA克隆，并成功地利用基于S.A.AR86的表达系统进行体外和体内实验。c）初步数据证明了外周接种后S.A.AR86对小鼠骨和关节组织的急性嗜性，并在这些组织中持续至少3个月。d）与辛德比斯组甲病毒的其他成员不同，S.A.AR86在所有年龄段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)鉴定S.A.AR86在骨/关节组织内复制的细胞靶点，表征S.A.AR86在这些组织内的持久性，并评价其他关节炎/关节痛相关披膜病毒（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)将通过将S.A.AR86基因组内的这些密码子改变为非神经毒力病毒中发现的密码子并筛选神经毒力丧失，评价非结构基因内神经毒力的9个推定遗传决定因素。 此外，将候选S.A.AR86密码子引入非神经毒性病毒基因组中，并筛选毒力增益。3)突变调节成年小鼠神经毒力的机制将使用目标2中鉴定的非结构基因突变体以及已经鉴定、克隆和部分表征的nsP 1 538突变进行检查。

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.