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: 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
• 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' 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

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.

总结 这个多PI项目的长期目标是确定宿主-病原体相互作用是如何 脑炎甲病毒在中枢神经系统中的影响进入、感染和传播 （CNS）。媒介传播的嗜神经病毒，委内瑞拉，东部和西部马 脑炎病毒（VEEV、EEEV、WEEV）在皮下接种后侵入CNS， 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、成骨细胞和其它细胞类型（EEEV、WEEV）。EEEV和VEEV 通过血行途径进入大脑，但只有VEEV在嗅觉神经元中被发现。的 CNS缺乏实质内淋巴组织，不能启动适应性免疫应答; 因此，它主要依赖于通过局部和全身细胞因子传递的先天反应 分泌，调节驻留神经细胞的状态并限制病毒的神经侵袭， 感染的程度。CNS驻留细胞可以包括神经血管的多个成员。 神经元可以是神经元单位（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，可能直接感染或作用于区域或 全身产生的细胞因子。VEEV是一种高度嗜淋巴细胞的病毒， 外周接种后的细胞因子反应，而淋巴组织中的EEEV嗜性是 高度受限，血清细胞因子应答低得多，在I型IFN的情况下， 往往无法察觉。在已发表的研究中，EEEV在人类和 小鼠模型定义了抑制免疫前哨髓样细胞复制的机制， 细胞，并极大地限制了体外对EEEV感染的先天性免疫（尤其是干扰素）应答 和在体内1.这些因素，其中包括存在的结合位点， EEEV 3'非翻译区（UTR）1-3中的造血细胞特异性microRNA，mir 142 - 3 p， EEEV与细胞上硫酸乙酰肝素（HS）受体的有效结合4，5， EEEV与VEEV的神经侵袭和CNS播散的差异尚不清楚。 支持EEEV与VEEV细胞因子诱导的差异可能对 神经侵袭，我们发现I型IFN依赖性反应直接调节转胞吞作用， 防止甲病毒进入血脑屏障并调节感染和损伤的水平， NVU 6的细胞。因此，甲病毒感染期间的全身和局部细胞因子应答 诱导BMEC和周细胞调节病毒进入血脑屏障（BBB）， 可能是其他CNS部位。这与病毒复制的相对程度一致， EEEV表现出广泛的神经元感染， CNS，而VEEV复制更集中（未发表）。使用突变的VEEV和EEEV， 表达感染指示物的新型病毒载体（例如，eGFP，纳米荧光素酶），我们 已经观察到这些甲病毒之间主要进入位点的区域异质性。 关于血脑屏障的进入，我们的研究也表明病毒的神经侵袭先于血脑屏障 破坏，利用小窝蛋白介导的转胞吞作用穿过BBB。我们假设 I型干扰素应答对EEEV和VEEV的进入和感染的影响不同， NVU通过病毒特异性诱导复制限制性先天免疫应答。 为了验证这些假设，我们将： 目标1.定义调节病毒进入的甲病毒和宿主特异性体外机制 以及NVU的感染 目的2：确定I型IFN在保护免受甲病毒感染中的体内功能作用 神经侵入的研究