Viral and host factors in neuroinvasion of encephalitis alphaviruses

脑炎甲病毒神经侵袭的病毒和宿主因素

• 批准号: 10659110
• 负责人: WILLIAM B KLIMSTRA
• 金额: $ 61.66万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659110
• 关键词: 3' Untranslated Regions; Alphavirus; Alphavirus Infections; Area; Astrocytes; Binding; Binding Sites; Biological Warfare; Bioterrorism; Blood - brain barrier anatomy; Brain; Brain imaging; Category B pathogen; Caveolins; Cells; Central Nervous System; Cessation of life; Communication; Dendritic Cells; Disease; Disease Outbreaks; Eastern Equine Encephalomyelitis; Encephalitis; Exhibits; Future; Hematogenous; Hematopoietic; Heparitin Sulfate; Heterogeneity; Human; Human Activities; IFNAR1 gene; Imaging Techniques; Immune; Immunoelectron Microscopy; In Vitro; Incidence; Infection; Injury; Innate Immune Response; Integration Host Factors; Interferon Type I; Interferons; Intranasal Administration; Invaded; Latin America; Lymphoid Tissue; Macrophage; Maps; Mediating; MicroRNAs; Microglia; Modeling; Myeloid Cells; Neurons; New England; Pericytes; Peripheral; Phenotype; Publishing; Registries; Role; Route; Sentinel; Serum; Signal Transduction; Site; South America; Testing; Therapeutic; Tropism; United States; United States National Institutes of Health; Venezuelan; Venezuelan Equine Encephalitis Virus; Viral; Viral Vector; Virulence; Virulence Factors; Virus; Virus Replication; Western Equine Encephalitis Virus; adaptive immune response; blood-brain barrier crossing; blood-brain barrier disruption; brain cell; brain endothelial cell; cell type; climate change; climate impact; cytokine; experimental study; genomic locus; gulf coast; human model; in vivo; interferon alpha receptor; member; mortality; mouse model; mutant; nanoluciferase; neurotropic virus; neurovascular unit; novel; pathogen; prevent; receptor; response; subcutaneous; trafficking; transcytosis; ultra high resolution; vector mosquito; vector-borne

SUMMARY The long-term objective of this multi-PI project is to determine how host-pathogen interactions impact entry, infection, and spread of encephalitic alphaviruses in the central nervous system (CNS). The vector-borne neurotropic viruses, Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, WEEV), invade the CNS after subcutaneous inoculation and initial interaction with immune sentinel cells, such as macrophages and dendritic cells (DCs) (VEEV), or fibroblastic, osteoblastic and other cell types (EEEV, WEEV). Both EEEV and VEEV enter the brain via the hematogenous route but only VEEV is found in olfactory neurons. The CNS lacks intraparenchymal lymphoid tissues and cannot initiate adaptive immune responses; thus, it mainly relies on innate responses communicated through local and systemic cytokine secretion, which modulate the status of resident neural cells and limits viral neuroinvasion and the extent of infection. CNSresident cells may include multiple members of the neurovascular unit (NVU) such as brain microvascular endothelial cells (BMECs), pericytes, astrocytes, microglia and neurons themselves that may be infected directly or acted upon by regionally or systemically produced cytokines. VEEV is a highly lymphotropic virus eliciting robust serum cytokine responses after peripheral inoculation, while EEEV tropism in lymphoid tissues is highly restricted, and serum cytokine responses are much lower, and in the case of type I IFN, often undetectable. In published studies, two primary virulence factors for EEEV in human and murine models defined mechanisms that suppresses replication in immune sentinel myeloid cells and greatly limit innate immune (especially interferon) responses to EEEV infection in vitro and in vivo1. How these factors, which include the presence of binding sites for the hematopoietic cell-specific microRNA, mir142-3p, in the EEEV 3' untranslated region (UTR)1-3, and efficient binding of EEEV to heparan sulfate (HS) receptors on cells4,5, impact the differential neuroinvasion and CNS dissemination of EEEV versus VEEV is unknown. Supporting the idea that differences in EEEV versus VEEV cytokine induction may be critical to neuroinvasion, we found that type I IFN-dependent responses directly regulate transcytosis, preventing alphavirus entry across the BBB and modulating the level of infection and injury in cells of the NVU6. Thus, systemic and local cytokine responses during alphavirus infection induce BMECs and pericytes to regulate viral entry at the blood-brain barrier (BBB) and potentially other CNS sites. This is consistent with the relative extent of virus replication at terminal stages of disease as EEEV exhibits widespread infection of neurons throughout the CNS while VEEV replication is much more focal (unpublished). Using mutant VEEV and EEEV, novel viral vectors that express indicators of infection (e.g., eGFP, nanoLuciferase) in vivo, we have observed regional heterogeneity in dominant sites of entry between these alphaviruses. With regard to BBB entry, our studies also indicate that viral neuroinvasion precedes BBB disruption, utilizing caveolin-mediated transcytosis to cross the BBB. We hypothesize that type I interferon responses differentially impact the entry and infection of EEEV and VEEV at the NVU via virus-specific induction of replication-restricting innate immune responses. To test these hypotheses we will: Aim 1. Define alphavirus and host specific mechanism in vitro that regulate viral entry and infection at the NVU. Aim 2: Define the in vivo functional role of type I IFN in protection from alphavirus neuroinvasion at the NVU.

摘要 这个多PI项目的长期目标是确定宿主和病原体如何相互作用 脑型甲型病毒在中枢神经系统的侵入、感染和传播 (CNS)。媒介传播的嗜神经性病毒，委内瑞拉、东部和西部的马 脑炎病毒(VEEV，EEEV，WeEV)，皮下接种后侵入中枢神经系统， 与免疫哨兵细胞，如巨噬细胞和树突状细胞(DC)的初始相互作用 (Veev)，或成纤维细胞、成骨细胞和其他细胞类型(EEEV、Weev)。EEEV和VEEV 通过血源性途径进入大脑，但在嗅觉神经元中只发现VEEV。这个 中枢神经系统缺乏实质内淋巴组织，不能启动适应性免疫反应； 因此，它主要依赖于通过局部和系统细胞因子传递的先天反应。 分泌，它调节驻留神经细胞的状态，限制病毒的神经侵袭和 感染的程度。CNS驻留细胞可能包括神经血管的多个成员 单位(NVU)，如脑微血管内皮细胞、周细胞、星形胶质细胞、 小胶质细胞和神经元本身可能直接感染或作用于区域性或 系统产生的细胞因子。Veev是一种高度嗜淋巴的病毒，能产生强健的血清。 外周接种后细胞因子反应，而淋巴组织中EEEV的趋向性 高度限制性，而且血清细胞因子反应要低得多，在I型干扰素的情况下， 通常是不可察觉的。在已发表的研究中，两个主要的EEEV毒力因素在人类和 小鼠模型确定了抑制免疫前哨髓系复制的机制 细胞和极大地限制先天性免疫(特别是干扰素)对EEEV感染的体外反应 在活生生的第一天。这些因素，其中包括结合位点的存在如何 造血细胞特异性microRNA，mir142-3p，位于EEEV 3‘非翻译区(UTR)1-3， 和EEEV与细胞4，5上的硫酸乙酰肝素(HS)受体的有效结合，影响 EEEV和VEEV的不同神经侵袭和中枢神经系统播散尚不清楚。 支持EEEV和VEEV细胞因子诱导的差异可能是 神经侵袭，我们发现I型干扰素依赖的反应直接调节细胞转运， 防止甲型病毒通过血脑屏障进入，并调节感染和损伤的水平 NVU6的细胞。因此，甲型病毒感染期间的全身和局部细胞因子反应 诱导BMEC和周细胞调节病毒进入血脑屏障(BBB)和 可能是其他CNS网站。这与病毒复制的相对范围在 疾病的终末期，因为EEEV表现出广泛的神经元感染 CNS，而VEEV复制则更有针对性(未发表)。使用突变的VEEV和EEEV， 在体内表达感染指示物(例如，绿色荧光蛋白、纳米荧光素酶)的新型病毒载体 在这些甲型病毒之间的优势进入部位观察到了区域异质性。 关于血脑屏障的进入，我们的研究还表明，病毒神经侵袭先于血脑屏障 破坏，利用小窝蛋白介导的细胞穿透血脑屏障。我们假设这种类型 I干扰素应答对EEEV和VEEV进入和感染的不同影响 NVU通过病毒特异性诱导限制复制的先天免疫反应。 为了检验这些假设，我们将： 目的1.确定甲型病毒和宿主特异性的体外机制，以调节病毒进入 以及新弗吉尼亚大学的感染。 目的2：明确I型干扰素在体内对甲型病毒的保护作用 北卡罗来纳大学的神经入侵。