Identifying Factors Limiting CHIKV Disease by Global Transcriptional Profiling

项目总结(参见说明) ：基孔肯雅病毒(CHIKV)在人类中引起蚊媒疾病的爆炸性流行，患有急性发热性疾病和严重的持续性多发性关节炎。既没有获得许可的疫苗，也没有有效的疗法来保护高危人群。在我们的关节相关CHIKV疾病的小鼠模型中，野生型CHIKV La ReUnion(CHIKV-LR)株引起肌肉骨骼炎症，而在感染美国陆军开发的实验性减毒活疫苗(181/25)的小鼠中没有观察到疾病。CHIKV致病、减毒和免疫原性的机制尚未得到很好的描述，但可能与干扰素的敏感性有关。干扰素系统，如果在没有病毒拮抗作用的情况下被激活，在动物模型中非常有效地阻止甲型病毒的复制，并显著改善疾病。尽管CHIKV-LR感染细胞中抗病毒状态的建立受到抑制，但当干扰素-a/B和干扰素-γ的反应减弱时，关节炎在体内会加剧，这意味着疾病控制至少部分是由于这个系统。然而，181/25在宿主对感染反应的其他方面也可能发生改变。树突状细胞(DO)、单核/巨噬细胞(MO/Mphi)和成骨细胞(OB)与CHIKV关节炎密切相关，在体外，如果在CHIKV感染前这些细胞中建立了干扰素介导的抗病毒状态，病毒复制就会受到抑制。我们建议在这些研究中解决三个主要问题：i)哪些干扰素刺激的效应器在抑制野生型CHIKV-LR方面有效，它们是通过什么机制发挥作用的(S)；ii)181/25疫苗株对这些效应器更敏感；iii)宿主细胞反应谱是否区分疫苗和野生型病毒感染？在目标1中，我们将与Katze实验室(华盛顿大学)合作，使用系统生物学方法识别这些细胞类型中受干扰素-a/B和/或干扰素-γ转录调控并具有潜在抗野生型CHIKV活性的基因。消减方法将被用来消除直接受病毒调控的基因，这些基因不太可能是抗病毒的，因为它们不能保护细胞。然后，我们将在干扰素诱导或未诱导的细胞中通过靶向siRNA敲除来测试候选效应器对CHIKV-LR和181/25毒株的抗病毒活性。随后，在目标2中，我们将研究通过在可诱导细胞系中过度表达已证实的效应物来抑制病毒的点。这也将确定181/25病毒是否表现出对干扰素系统效应器的诱导或敏感性增强。最后，在目标3中，将挖掘数据，以确定是否存在与宿主对疫苗的反应相关的、可与野生型病毒区分的感染的“特征”，例如炎症反应基因、MO/Mphi激活和迁移因子的调节改变，或细胞应激反应。如提案所述，将通过协作、数据和试剂共享建立与现有PNWRCE项目/调查人员的协同作用。

PROJECT SUMMARY (See instructions) : Chikungunya virus (CHIKV) causes explosive epidemics of mosquito-borne disease in humans, with acute febrile illness and crippling, persistent polyarthritis. Neither licensed vaccines nor effective therapies are available to protect at-risk populations. In our mouse model of joint-associated CHIKV disease, the wild-type CHIKV La Reunion (CHIKV-LR) strain caused musculoskeletal inflammatory disease while no disease was observed in mice infected with an experimental live-attenuated vaccine developed by the U.S. Army (181/25). Mechanisms underlying CHIKV pathogenesis, attenuation and immunogenicity are not well characterized, but may be linked to interferon (IFN) sensitivity. The IFN system, if activated in the absence of virus antagonism, is very effective at blocking the replication of alphaviruses and substantially ameliorating disease in animal models. Although establishment of the antiviral state is suppressed in CHIKV-LR-infected cells, arthritic disease is exacerbated In vivo when IFN-a/B and IFN-y responses are diminished, implying that disease control is at least partially due to this system. However, 181/25 may also be altered in other aspects of the host response to infection. Dendritic cells (DO), monocyte/macrophages (MO/Mphi) and osteoblasts (OB) are highly relevant to CHIKV arthritic disease and, in vitro, if an IFN-mediated antiviral state is established in these cells before CHIKV infection, virus replication is curtailed. We propose to address three major questions in these studies: i) which IFN-stimulated effectors are effective in inhibiting wild-type CHIKV-LR and by what mechanism(s) do they function; ii) is the 181/25 vaccine strain more sensitive to these effectors; and iii) do host cell response profiles distinguish vaccine from wild-type virus infections? In Aim 1, we will collaborate with the Katze Laboratory (University of Washington) using a systems biology approach to identify genes in these cell-types that are transcriptionally regulated by IFN-a/B and/or IFN-y and have potential antiviral activity against wild type CHIKV. A subtractive approach will be used to eliminate genes that are directly virus-regulated and unlikely to be antiviral as they do not protect cells. We will then test the antiviral activity of candidate effectors versus CHIKV-LR and 181/25 strains by targeted siRNA knockdown in IFN-primed or unprimed cells. Subsequently in Aim 2, we will study the point of virus inhibition by over-expression of confirmed effectors in inducible cell lines. This will also determine if the 181/25 virus exhibits enhanced induction of or sensitivity to effectors of the IFN system. Finally, in Aim 3, data will be mined to determine if there are "signatures" of infection associated with the host response to the vaccine that can be distinguished from the wild-type virus, such as altered regulation of inflammatory response genes, MO/Mphi activation and migration factors, or cell stress responses. Synergy with existing PNWRCE projects/ investigators will be established through collaboration, data and reagent sharing, as outlined in the proposal.