TOGAVIRUS TROPISM FOR BONES, JOINTS, AND CNS

人类TogaVirus感染的特征是急性和持续性的关节炎/关节痛，以及病毒性脑炎。成年小鼠感染S.A.AR86是Togaviridae家族的一种甲型病毒，为研究这两种人类疾病的分子基础提供了一个极好的和独特的模型系统。这些模型的吸引人的特征包括：a)S.A.AR86和相关病毒导致人类急性和持续性关节痛。B)我们已经对S.A.AR86及其近亲的基因组进行了测序，构建了一个真实的S.A.AR86感染性克隆，并成功地利用基于S.A.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周围接种并持续至少3个月后，S.A.AR86对这些组织具有急性趋向性。D)与甲型病毒Sindbis组的其他成员不同，S.A.AR86在所有年龄段的小鼠中都是神经毒力的，并且这种表型映射到负责RNA合成的病毒非结构基因。我们提出了以下具体目标。1)确定S.A.AR86在骨/关节组织中复制的细胞靶点，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TOGA病毒，包括风疹和罗斯河病毒，以确定其在骨/关节组织中的复制和/或持久性。2)通过将S.A.AR86基因组中的这些密码子改变为在非神经毒力病毒中发现的密码子并筛选神经毒力的丧失，将评估非结构基因中9个可能的神经毒力遗传决定因素。此外，候选的S.A.AR86密码子将被引入到非神经毒力病毒基因组中，并筛选毒力的获得。3)突变调节成年小鼠神经毒力的机制(S)将利用AIM 2中确定的非结构基因突变以及已被鉴定、克隆和部分表征的nsP1538突变来进行研究。

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.