Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection
Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection
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DOI：
10.1128/jvi.03219-15
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发表时间：
2016-06-01
影响因子：
5.4
通讯作者：
Olson, Ken E.
中科院分区：
文献类型：
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作者：
Phillips, Aaron T.;Rico, Amber B.;Olson, Ken E.
委内瑞拉和西部马脑炎病毒（VEEV和WEEV;甲病毒属;披膜病毒科）是蚊媒病原体，可引起人类和马科动物的中枢神经系统（CNS）疾病。成年CD-1小鼠在感染VEEV和WEEV后也发生CNS疾病。通过鼻内（i. n.）通过嗅觉神经元（OSNs）进入脑内。在这项研究中，我们用表达萤火虫荧光素酶（fLUC）的重组WEEV（McMillan）或VEEV（亚型IC毒株3908）注射小鼠足垫，以模拟蚊子感染，并检查甲病毒进入CNS。荧光素酶表达作为感染的标志物，通过体内和离体成像检测为生物发光（BLM）。BLM成像检测WEEV和VEEV在接种后12小时（hpi）在注射部位（脚垫）和早在72 hpi在大脑中。来自WEEV的BLM。McM-fLUC和VEEV。3908-fLUC注射最初在脑的脑室周围器官（CVO）中检测到。在嗅神经上皮或OSNs中未检测到BLM活性。还在小鼠足垫中注射表达DsRed（Discosoma sp.）并通过共焦荧光显微镜成像。DsRed成像通过检测CVO中的WEEV，支持我们的BLM发现，该WEEV在沿神经元轴沿着扩散到其他脑区之前。综上所述，这些发现支持我们的假设，即外周注射的甲病毒通过CVO的血行接种进入CNS，然后沿神经元轴沿着向心传播。重要的是，VEEV和WEEV是在美洲引起散发性流行病的蚊媒病毒。在马、人和小鼠感染模型中，这两种病毒均与CNS疾病相关。在这项研究中，我们将VEEV或WEEV注射到远系繁殖的CD-1小鼠的足垫中，以模拟蚊子的传播，这些VEEV或WEEV被设计为表达生物发光或荧光报告基因（分别为fLUC和DsRed）。报告基因表达可作为VEEV和WEEV复制和感染的可检测生物发光和荧光标记。使用生物发光成像、组织学检查和共聚焦荧光显微镜来鉴定这些甲病毒在CNS中的早期进入位点。我们观察到，大脑的特定区域（脑室周围器官[CVO]）始终显示出VEEV和WEEV感染的最早体征。组织学检查支持VEEV和WEEV在血脑屏障天然缺失的特定部位进入小鼠脑。
Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV. McM-fLUC and VEEV. 3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis.IMPORTANCEVEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.