Pathogenesis of Tick-Borne Flavivirus Infections

蜱传黄病毒感染的发病机制

• 批准号: 9566630
• 负责人: Marshall Elliott Bloom
• 金额: $ 74.31万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9566630
• 关键词: Acute; Adult; Alkhurma Virus; Antibody Response; Antiviral Agents; Apoptosis; Arthropods; Attenuated; Basic Science; Bioinformatics; Biology; Black-legged Tick; C57BL/6 Mouse; CASP3 gene; Cell Death; Cell Line; Cell Survival; Cells; Cellular Membrane; Cellular biology; Computer software; Confocal Microscopy; Culicidae; Deer Tick; Dengue Virus; Disease; Electron Microscopy; Encephalitis; Endoplasmic Reticulum; Experimental Models; Family; Fever; Flavivirus; Flavivirus Infections; Genes; Genomics; Human; Image; Imaging Techniques; Immunology; In Vitro; Inbred BALB C Mice; Infection; Japanese encephalitis virus; Kyasanur Forest disease virus; Laboratories; Langat virus; Latin America; Lead; Life Cycle Stages; Lytic; Maintenance; Mammalian Cell; Meningitis; Modeling; Molecular; Molecular Biology; Molecular Virology; Mus; Neurologic; Omsk hemorrhagic fever virus; Pathogenesis; Pathway Analysis; Pathway interactions; Peromyscus; Phase; Play; Publications; Publishing; Research; Resolution; Rodent; Role; Seminal fluid; Site; Staining method; Stains; Survivors; Syndrome; System; Tick-Borne Encephalitis Virus; Tick-Borne Encephalitis Viruses; Ticks; Time; Vesicle; Viral; Viral Hemorrhagic Fevers; Viremia; Virulent; Virus; Virus Diseases; Virus Replication; West Nile virus; Work; Yellow fever virus; Zika Virus; deep sequencing; electron tomography; embryo cell; hemorrhagic fever virus; insight; intraperitoneal; member; neuroblastoma cell; neutralizing antibody; novel; olfactory bulb; pandemic disease; particle; pathogen; research study; transcriptome; transcriptomics; vector; virology

Vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). The dramatic and ongoing pandemic attributed to the MBFV Zika virus is important because of its capacity to cause a severe congenital Zika disease as well an ever increasing spectrum of neurological syndromes in adults. Our current research is focused on the TBFV, but studying the biology of TBFV will elucidate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We primarily study LGTV, a naturally attenuated member of the TBFV that can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. In addition, we also study the virulent autochthonous BSL-3 POWV/DTV. Neither of these 2 agents are Select Agents which greatly facilitate research studies. In addition, we have also begun to study the BSL2 MBFV, Zika virus. With the recent emergence of Zika in Latin America and the US, similar avenues of inquiry are being explored for that vexatious pathogen. Comparison of VBFV cytoarchitecture in mammalian and arthropod cells. We previously published a study comparing LGTV virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. This last year, we published similar studies with ZIKV in a human neuroblastoma cell line (SK-N-SH) and mosquito cell line C6/36 from A. albopictus. ZIKV replicated well in SK-N-SH and C6/36. ZIKV caused an acute lytic crisis accompanied by frank apoptosis in SK-N-SH cells, but the effects of infection in C6/36 cells were imperceptible despite the fact that activated caspase 3 staining was readily apparent. These findings mirrored those we have seen with the TBFV. The results of ultrastructural studies on SK-N-SH and C6/36 cells revealed that the cytoarchitecture of ZIKV infection was similar to that seen previously with the TBFV. However, the application of dual-tilt electron tomography lead to resolution substantially better than our previous work. Zika virus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. Differences between infected SK-N-SH and C6/36 were less prominent than those seen with TBFV. A Molecular biology and molecular pathogenesis of acute and persistent VBFV infection. The role of persistent infection in natural life cycle of TBFV in rodent and arthropod hosts is not well characterized, but may also be responsible for prolonged debilitating sequelae observed in survivors of acute TBFV infection. Experimental work by others on MBFV and the recent findings that ZIKV can persist in human semen and other sites for extended periods of time strongly imply that persistence of MBFV may also be an underappreciated feature of those infections. In the past year, we published our studies of TBFV persistence to inspect the cellular transcriptome during the initiation and maintenance of persistent infection. As noted, LGTV infection causes an acute lytic crisis of most cells, and we have shown the mechanism of cell death is apoptosis. The surviving cells somehow evade apoptosis and go on to establish persistent infection. Our agnostic deep sequencing approach to the study of cellular transcriptomics confirmed that gene pathways associated with cell survival and apoptosis avoidance were involved. A signature of 451 genes was associated with the initiation of persistence. Intensive bioinformatics perusal using Ingenuity Pathway Analysis and other similar software packages, revealed that networks associated with cell survival were playing a role. The acute phase was associated with networks and pathways. We also unpublished our novel deep sequencing approach to examine the viral genomics during TBFV infection in tick cells (ISE-6). ISE-6 cells develop a persistent infection with no apparent acute phase and there is no evidence of DI particles being involved in the initiation or maintenance of persistence. A transcriptomic analysis of infected ISE6 cells is being prepared for publication. TBFV infection in the reservoir host. Peromyscus leucopus is the identified reservoir host for POWV. Parallel POWV infection of Permomyscus and Balb/C and C57Bl/6 mice yielded surprising results which we published this year. POWV causes a fulminant and lethal neurological infection in both strains of lab mice after either intracerebral or intraperitoneal infection. However, the Peromyscus mice show no overt signs of disease after either ic or ip inoculation. Detailed studies reveal that no evidence of POWV infection can be detected after ip inoculation of Peromyscus mice and, furthermore that after ic infection, virus replication is severely restricted in time and place to the olfactory bulb and projecting tracts. Only a short-lived and very low level viremia occurs but a neutralizing antibody response does develop. The mechanism for the virus restriction is being pursued in vitro and in commercially available mouse embryo cell lines from B6 and Peromyscus.

属于Flaviviridae家族的Flaviviruse载体，属于Flaviviridae属Flavivivirus属，构成了一些最重要的新兴和重新出现的病毒病原体。 tick传播的黄病毒（TBFV）包括tick虫脑炎病毒（TBEV），OMSK出血性发烧病毒，Kyasanur Forest病毒，藻类性出血病毒，Powassan/deer tick tick病毒（POWV/DTV）和Langat Virus（LGTV）（LGTV）。 TBFV通常通过ixodid tick传递给人类，并引起各种疾病，从轻度的发热疾病到脑炎，脑膜炎或出血性发烧。蚊子传播的黄病毒包括西尼罗河病毒（WNV），日本脑炎病毒（JEV），登革热病毒（DEN）和黄热病病毒（YFV）。归因于MBFV寨卡病毒的戏剧性和持续的大流行很重要，因为它有能力引起严重的先天性寨卡病毒，并且成人神经综合症的频谱越来越多。 我们目前的研究集中在TBFV上，但是研究TBFV的生物学将阐明其他载体传播病毒的生物学。我们实验室的研究采用病毒学，免疫学，高级成像技术，基因组学，细胞生物学，分子生物学和载体生物学。我们主要研究LGTV，这是TBFV的自然减弱成员，可以在BioSafety Level-2（BSL-2）上进行安全研究，这使其成为深入了解TBFV的绝佳模型。此外，我们还研究了有毒的自围Hon-3 POWV/DTV。这两种代理都不是精选的代理，极大地促进了研究。此外，我们还开始研究BSL2 MBFV，寨卡病毒。随着拉丁美洲和美国寨卡病毒最近出现的出现，这种令人烦恼的病原体正在探索类似的询问途径。 哺乳动物和节肢动物细胞中VBFV细胞结构的比较。我们先前发表了一项研究，比较了利用分子病毒学以及共聚焦显微镜，电子显微镜和电子断层扫描的LGTV病毒感染和tick细胞系中的LGTV病毒感染。去年，我们在人类神经细胞瘤细胞系（SK-N-SH）和蚊子细胞系C6/36中发表了类似的ZIKV研究。 ZIKV在SK-N-SH和C6/36中很好地复制了。 ZIKV引起了急性裂解危机，并伴有SK-N-SH细胞中坦率的细胞凋亡，但是尽管很明显地显然表明了激活的caspase 3染色，但C6/36细胞中感染的作用是不可察觉的。这些发现反映了我们在TBFV中看到的发现。 关于SK-N-SH和C6/36细胞的超微结构研究的结果表明，ZIKV感染的细胞结构与以前在TBFV中看到的相似。但是，双倾斜电子断层扫描的应用可使分辨率大大好于我们以前的工作。哺乳动物细胞系中的寨卡病毒感染伴随着大量的增殖和细胞膜重排，主要来自内质网。电子断层扫描显示病毒引起的球形囊泡被认为可以保护复制中间体免受细胞内抗病毒系统的影响。受感染的SK-N-SH和C6/36之间的差异不如TBFV看到的差异。一个 急性和持续性VBFV感染的分子生物学和分子发病机理。持续感染在TBFV自然生命周期中在啮齿动物和节肢动物宿主中的作用尚未很好地表征，但也可能导致在急性TBFV感染的幸存者中延长衰弱的后遗症。其他人在MBFV上的实验性工作以及最近的发现，ZIKV可以在人类精液和其他部位长期持续存在很长时间，这强烈暗示MBFV的持久性也可能是这些感染的不足特征。 在过去的一年中，我们发表了有关TBFV持久性的研究，以检查持续感染的启动和维持过程中的细胞转录组。如前所述，LGTV感染会导致大多数细胞的急性裂解危机，我们已经表明细胞死亡的机理是凋亡。幸存的细胞以某种方式逃避了凋亡，并继续建立持续的感染。我们对细胞转录组学研究的不可知论深度测序方法证实，与细胞存活和避免凋亡相关的基因途径涉及。 451个基因的签名与持久性的启动有关。使用Ingenuity途径分析和其他类似软件包的密集生物信息学细胞表明，与细胞存活相关的网络起着作用。急性相与网络和途径有关。 我们还未发表我们的新型深层测序方法，以检查tbick细胞中TBFV感染期间病毒基因组学（ISE-6）。 ISE-6细胞形成持续感染，没有明显的急性相，并且没有证据表明DI颗粒参与持续性的启动或维持。正在准备对受感染的ISE6细胞进行转录组分析以发表。 水库主机中的TBFV感染。 Peromyscus leucopus是POWV的确定储层宿主。固定库和BALB/C和C57BL/6小鼠的平行POWV感染产生了令人惊讶的结果。 POWV在脑内或腹膜内感染后引起两种实验室小鼠菌株的暴发性和致命神经系统感染。但是，Peromyscus小鼠在IC或IP接种后没有明显的疾病迹象。详细的研究表明，在IP接种Peromyscus小鼠后无法检测到POWV感染的证据，此外，在IC感染后，病毒复制在时间和地点严重限制到嗅球和投射区。仅发生短暂且非常低的病毒血症，但确实会发展中和抗体反应。病毒限制的机制在体外和来自B6和Peromyscus的市售小鼠胚胎细胞系中被追求。