Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes
Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes
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DOI：
10.1128/jvi.01682-21
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发表时间：
2022-01-01
影响因子：
5.4
通讯作者：
Mackow, Erich R.
中科院分区：
文献类型：
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作者：
Conde, Jonas N.;Sanchez-Vicente, Santiago;Mackow, Erich R.
鲍瓦桑病毒(Powassan Virus，POWV)是一种由硬蜱传播的神经致死性黄病毒，在美国东北部出现，在长岛(Li)鹿蜱中的流行率为2%。被扁虱叮咬15分钟内传播的病毒会进入中枢神经系统(CNS)，导致脑炎(10%的病例是致命的)和长期的神经元损伤。用硬蜱匀浆直接接种VeroE6细胞，免疫过氧化物酶染色检测感染POWV的细胞，分离出存在于硬蜱体内的POWV-LI9和POWV-LI41。接种的POWV-LI9和LI41仅存在于感染的细胞灶中，表明细胞间的扩散，尽管在液体培养中没有覆盖物生长。克隆和测序证实了POWV-LI9是在鹿体内传播的一种不同的II系POWV毒株。原代人脑微血管内皮细胞(HBMECs)和周细胞形成神经血管复合体，限制其进入中枢神经系统。我们发现POWV-LI9和-LI41和Lineage I POWV-LB能有效地感染hBMECs和周细胞，并且POWV可以从hBMECs向下室周细胞传递，而不渗透极化的hBMECs。POWV-LI9同步感染人骨髓微血管内皮细胞和周细胞可诱导促炎性趋化因子、干扰素-β和干扰素刺激基因家族蛋白的表达，使感染的周细胞延迟分泌干扰素-β。干扰素抑制了POWV的感染，但尽管有干扰素的分泌，POWV感染的hBMEC和周细胞的一部分仍然持续感染。这些发现提示了POWV(LI9/LI41和LB)感染hBMEC，向基底部扩散到周细胞，并进入中枢神经系统的潜在机制。人血红细胞微血管内皮细胞和周细胞对POWV感染的反应提示了POWV神经毒力的免疫病理学作用和防止POWV扩散到神经室的潜在治疗靶点。值得注意的是，用含有POWV的硬蜱匀浆接种VeroE6细胞后，在液体培养中出现了感染细胞的病灶，这与细胞间的传播一致。POWV-LI9、-LI41和POWV-LB株感染hBMECs和构成神经血管复合体的周细胞。POWV从感染的人骨髓微血管内皮细胞非裂解地传播到下室周细胞，提示了POWV通过血脑屏障(BBB)传播的机制。POWV-LI9可诱导感染的hBMEC和周细胞的炎症反应，这可能与免疫细胞募集和神经发病有关。这项研究揭示了POWV通过感染人骨髓微血管内皮细胞并向基底部扩散到腔周细胞而进入中枢神经系统的潜在机制。我们的发现表明，POWV-LI9持续存在于形成跨越血脑屏障的神经血管复合体的细胞中，并提出了防止POWV扩散到神经室的潜在治疗靶点。
Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-beta (IFN-beta) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-beta secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments.IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.