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病原体的相互作用如何中枢神经系统中脑甲状腺病毒的撞击进入，感染和传播（CNS）。媒介传播的神经性病毒，委内瑞拉，东部和西马脑炎病毒（VEEV，EEEV，WEEV），在接种和接种后侵入CNS 与免疫前哨细胞的初始相互作用，例如巨噬细胞和树突状细胞（DCS）（VEEV），或成纤维细胞，成骨细胞和其他细胞类型（EEEV，WEEV）。 EEEV和VEEV都通过血源性途径进入大脑，但在嗅觉神经元中仅发现VEEV。这中枢神经系统缺乏核内淋巴组织，无法引发适应性免疫反应。因此，它主要依赖于通过局部和系统性细胞因子传达的先天反应分泌，调节常驻神经细胞的状态并限制了病毒神经侵袭和感染程度。 CNSisentent细胞可能包括神经血管的多个成员单位（NVU），例如脑微血管内皮细胞（BMEC），周细胞，星形胶质细胞，小胶质细胞和神经元本身可能直接感染或通过区域或作用系统地产生细胞因子。 VEEV是一种高度淋巴的病毒，引起了鲁棒的血清周围接种后的细胞因子反应高度限制，血清细胞因子反应要低得多，在I型IFN的情况下，通常无法检测到。在发表的研究中，人类EEEV的两个主要毒力因素和鼠模型定义了抑制免疫前哨髓样复制的机制细胞并大大限制了先天免疫（尤其是干扰素）对EEEV感染的反应和invivo1。这些因素如何包括存在结合位点造血细胞特异性microRNA，miR142-3p，在EEEV 3'未翻译区（UTR）1-3中 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的差异神经入侵和中枢神经系统的传播是未知的。支持以下观点，即EEEV与VEEV细胞因子诱导的差异可能对神经侵入性，我们发现I型IFN依赖性反应直接调节转胞膜，防止α病毒进入BBB，并调节感染水平和伤害 NVU6的细胞。因此，α病毒感染期间的全身和局部细胞因子反应诱导BMEC和周细胞在血脑屏障（BBB）和潜在的其他CNS站点。这与病毒复制的相对程度一致疾病的终末阶段作为EEEV在整个过程中表现出广泛的神经元感染中枢神经系统虽然VEEV复制更为焦点（未发表）。使用突变体Veev和EEEV，在体内表达感染指标（例如EGFP，纳米酸酯酶）的新型病毒载体，我们已经观察到这些α病毒之间主要进入部位的区域异质性。关于BBB的进入，我们的研究还表明，病毒神经侵入之前破坏，利用小窝蛋白介导的转介症越过BBB。我们假设这种类型我干扰素的反应差异地影响了EEEV和VEEV的进入和感染通过病毒特异性诱导复制限制先天免疫反应的NVU。为了检验这些假设，我们将： AIM 1。定义α病毒和宿主在体外调节病毒的特定机制和NVU的感染。 AIM 2：定义I型IFN在保护α病毒中的体内功能作用 NVU的神经入侵。