Mechanism and Functional Impact of H5N1 Influenza Virus Replication in Macrophages

 描述（由申请方提供）：高致病性禽（HPAI）H5N1流感病毒与50%的人类死亡率相关，被认为是主要的大流行威胁。虽然这些病毒引起严重疾病的机制尚未完全确定，但过度攻击性的免疫反应是H5N1疾病的标志。巨噬细胞，虽然对保护免受流感病毒感染至关重要，但已涉及严重的H5N1疾病，主要是通过产生过量的促炎细胞因子。这项拟议项目的初步证据表明，HPAI H5N1流感病毒的一个定义特征是它们能够以血凝素（HA）依赖的方式在巨噬细胞中高效复制，而非H5流感病毒在病毒进入细胞质后会降解。此外，H5N1病毒在巨噬细胞中复制的能力与体内疾病的严重程度相关。目前还不清楚H5N1流感病毒如何破坏限制流感病毒在巨噬细胞中复制的细胞途径。该提案测试了这样的假设，即自噬的细胞途径通过靶向它们在溶酶体中降解来限制非H5流感病毒在巨噬细胞中的复制，而H5N1病毒不能激活自噬，从而能够完成病毒复制周期。此外，我们假设H5N1流感病毒在巨噬细胞中的复制改变了细胞功能。将使用亲代H1流感病毒和表达H5 HA蛋白的反向遗传衍生病毒（已取消选择，以允许其在BSL 2实验室条件下安全使用）来检验该假设。具体目标1测试自噬限制非H5流感病毒复制的模型。共聚焦成像和蛋白质印迹的组合将用于检测病毒感染期间的自噬激活。感染将在自噬抑制剂存在下进行，病毒复制将通过定量实时PCR测定。具体目标2检验巨噬细胞功能因H5N1病毒复制而受损的假设。将通过酶联免疫吸附试验（ELISA）测定促炎细胞因子和一氧化氮水平，并使用吞噬试验测定感染病毒（在该细胞类型中复制或不复制）的细胞中巨噬细胞功能的变化。该提案产生的数据不仅将增加我们对H5N1流感病毒如何导致过度疾病的理解，而且还将增加我们对微生物如何“劫持”巨噬细胞中的抗病毒途径以支持其自身复制的理解。因此，拟议的项目将可能对感染巨噬细胞的病原体的更广泛领域产生重要影响。

 DESCRIPTION (provided by applicant): Highly pathogenic avian (HPAI) H5N1 influenza viruses are associated with a 50% mortality rate in humans and are considered a primary pandemic threat. Although the mechanism by which these viruses cause severe disease is not fully defined, an overly aggressive immune response is a hallmark of H5N1 disease. Macrophages, though critical for protection against influenza virus infection, have been implicated in severe H5N1 disease primarily through the production of excessive levels of pro-inflammatory cytokines. The preliminary evidence for this proposed project demonstrates that a defining feature of HPAI H5N1 influenza viruses is their ability to productively replicate in macrophages in a hemagglutinin (HA)-dependent manner while non-H5 influenza viruses are degraded in the cytoplasm upon viral entry. Further, the ability of H5N1 viruses to replicate in macrophages is associated with disease severity in vivo. It is unclear how H5N1 influenza viruses subvert cellular pathways which restrict influenza virus replication in macrophages. This proposal tests the hypothesis that the cellular pathway of autophagy restricts non-H5 influenza virus replication in macrophages by targeting them for degradation in the lysosome while H5N1 viruses fail to activate autophagy, thus being enabled to complete the viral replication cycle. Further, we hypothesize that H5N1 influenza virus replication in macrophages alters cellular functions. The hypothesis will be tested using a parental H1 influenza virus and a reverse genetics-derived virus expressing an H5 HA protein that has been deselected to permit its safe use under BSL2 laboratory conditions. Specific Aim 1 tests the model that autophagy restricts non-H5 influenza virus replication. A combination of confocal imaging and western blots will be used to detect autophagy activation during virus infection. Infections will be carried out in the presence of autophagy inhibitors and virus replication will be determined by quantitative real-time PCR. Specific Aim 2 tests the hypothesis that macrophage function is impaired by replication of H5N1 viruses. Proinflammatory cytokine and nitric oxide levels will be determined by enzyme-linked immunosorbent assay (ELISA) and a phagocytosis assay will be used to determine changes in macrophage function in cells infected with viruses that do or do not replicate in that cell type. The data generated by this proposal will not only increase our understanding of how H5N1 influenza viruses cause excessive disease, but will also increase our understanding of how microbes "hijack" antiviral pathways in macrophages to support their own replication. Thus, the proposed project will potentially have an important impact on the broader field of pathogens that infect macrophages.