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

基本信息

批准号：
10389982
负责人：
WILLIAM B KLIMSTRA
金额：
$ 63.49万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/10389982
关键词：
3&apos 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 Cessation of life Dendritic Cells Disease Disease Outbreaks 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 Maps Mediating MicroRNAs Microglia Modeling Myeloid Cells Neuraxis 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 disruption brain cell brain endothelial cell cell type climate change cytokine experimental study genomic locus gulf coast human model in vivo macrophage member mortality mouse model mutant nanoluciferase neurotropic virus neurovascular unit novel pathogen prevent receptor response subcutaneous trafficking transcytosis vector mosquito vector-borne

项目摘要

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.

概括这个多PI项目的长期目标是确定宿主与病原体如何相互作用影响脑炎甲病毒在中枢神经系统的进入、感染和传播（中枢神经系统）。媒介传播的嗜神经病毒、委内瑞拉马、东部马和西部马脑炎病毒（VEEV、EEEV、WEEV）皮下接种后侵入中枢神经系统与免疫前哨细胞（例如巨噬细胞和树突细胞 (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 通过血行途径进入大脑，但仅在嗅觉神经元中发现 VEEV。这中枢神经系统缺乏实质内淋巴组织，无法启动适应性免疫反应；因此，它主要依赖于通过局部和全身细胞因子传达的先天反应分泌，调节驻留神经细胞的状态并限制病毒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脑微血管内皮细胞 (BMEC)、周细胞、星形胶质细胞、小胶质细胞和神经元本身可能被直接感染或通过区域或区域作用系统地产生细胞因子。 VEEV 是一种高度嗜淋巴细胞病毒，可产生强效血清外周接种后细胞因子反应，而淋巴组织中的 EEEV 趋向性高度受限，血清细胞因子反应要低得多，就 I 型 IFN 而言，常常无法检测到。在已发表的研究中，人类和动物中 EEEV 的两个主要毒力因子小鼠模型定义了抑制免疫前哨骨髓复制的机制细胞并极大地限制体外对 EEEV 感染的先天免疫（尤其是干扰素）反应和体内1。这些因素（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， EEEV 与细胞上硫酸乙酰肝素 (HS) 受体的有效结合4,5，影响 EEEV 与 VEEV 的神经侵袭和中枢神经系统传播差异尚不清楚。支持以下观点：EEEV 与 VEEV 细胞因子诱导的差异可能对于神经侵袭，我们发现 I 型 IFN 依赖性反应直接调节胞吞作用，防止甲病毒进入血脑屏障并调节感染和损伤水平 NVU6 的细胞。因此，甲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) 并潜在的其他中枢神经系统站点。这与病毒复制的相对程度一致 EEEV 在疾病的末期表现出神经元的广泛感染 CNS 而 VEEV 复制更为集中（未发表）。使用突变 VEEV 和 EEEV，在体内表达感染指标（例如，eGFP、纳米荧光素酶）的新型病毒载体，我们观察到这些甲病毒之间主要进入位点的区域异质性。关于 BBB 进入，我们的研究还表明病毒神经侵袭先于 BBB 破坏，利用小窝蛋白介导的转胞吞作用穿过血脑屏障。我们假设该类型 I 干扰素反应对 EEEV 和 VEEV 的进入和感染的影响存在差异 NVU 通过病毒特异性诱导复制限制的先天免疫反应。为了检验这些假设，我们将：目标 1. 定义甲病毒和调节病毒进入的体外宿主特异性机制以及 NVU 的感染。目标 2：确定 I 型 IFN 在预防甲病毒感染中的体内功能作用 NVU 的神经侵袭。