Pathogenesis of Tick-Borne Flavivirus Infections

蜱传黄病毒感染的发病机制

基本信息

批准号：
10927795
负责人：
Marshall Elliott Bloom
金额：
$ 159.19万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10927795
关键词：
Acinus organ component Adult Anaplasma Anaplasma phagocytophilum Animal Model Antiviral Agents Arthropods Artificial Membranes Attenuated Basic Science Binding Biological Assay Biology Black-legged Tick Blood Borrelia Borrelia burgdorferi Cells Cellular biology Central Nervous System Diseases Cessation of life Chronic Chronic Disease Collaborations Coxiella burnetii Digestion Disease Dissociation Entomology Experimental Models Flavivirus Infections Genes Genomics Human Image Imaging Techniques Immune response Immunology Infection Infectious Agent Integration Host Factors Laboratories Lyme Disease Methods Microbe Midgut Molecular Molecular Biology Mus Nymph Organ Culture Techniques Pathogenesis Pathway interactions Play Powassan virus Proliferating Proteomics Protocols documentation Publications Publishing Q Fever Refractory Research Rickettsia Rocky Mountain Spotted Fever Rodent Model Role Salivary Glands Side Supporting Cell Suspensions Symptoms System Testing Ticks Vaccine Design Vaccines Vial device Viral Virginia Virus Virus Diseases Virus Replication Virus-like particle Work acute infection animal model development cell type chronic infection co-infection deep sequencing feeding genome wide screen intestinal epithelium microorganism interaction mouse model nervous system disorder novel permissiveness receptor screening single cell analysis small molecule libraries therapy development tick feeding tick-borne tick-borne flavivirus tick-borne virus tool transmission process vector vector transmission vector-borne virology

项目摘要

Research in our laboratory employs virology, immunology, entomology, advanced imaging techniques, genomics, proteomics, cell biology, molecular biology, and vector biology. We study LGTV at BSL-2 and POWV/DTV at BSL-3. TBFV biology in I. scapularis. Infection in ticks is a critical, but understudied, feature of TBFV biology. Our ex vivo organ culture work has yielded considerable information about TBFV infection in midgut and salivary glands of adult ticks, and is presently under review for publication. In the past year, Dr. Ochwoto and Dr. Stewart employed artificial membrane blood feeding and demonstrated that adult I. scapularis ticks can be infected with relatively high efficiency by feeding them on LGTV infected blood. Studies on the midgut of fed adults revealed the expected differentiation and proliferation of the intestinal epithelium into mature digestive cells, and the results indicated that these cells support virus replication. Initial studies suggest that the salivary glands also become infected while the ticks are feeding and that prior infection of the midgut and subsequent systemic dissemination of virus are not required for infection of the salivary glands. The nymphal stage of the ticks is the most important for human infection, so in the past year we have started to study infection in nymphs. However, when efforts were made to infect them using the artificial membrane system, the results were disappointing and inconsistent. As a result, Dr. Weck is planning to infect nymphs by feeding them on LGTV infected mice. Salivary glands have at least 3 types of acini and each acini contains multiple cell types. It is unclear if all acinar types or all cell types are permissive for virus infection. One way to approach this interesting question would be to infect ex vivo salivary gland cultures, dissociate the infected cultures and subject the cells to single cell analysis. In the past year, Dr. Ochwoto and Ms. Theriault applied cell dissociation methods to dissected salivary glands from adult ticks. The acini were refractory to dissociation using simple enzymatic digestion and we have not yet obtained single cell suspensions satisfactory for analysis. Microbial interactions in Ixodes scapularis ticks. Ixodes scapularis ticks harbor multiple infectious agents, including TBFV, B. burgdorferi and Anaplasma phagocytophilum, but polymicrobial human infections are rare. A possible explanation is that infection with one microbe interferes with infection by a second. In the past year, Dr. Stewart established a collaboration with Dr Eastwood from Virginia Tech to examine co-infections with anaplasma and TBFV. Dr Stewart is also developing tools to identify the TBFV receptor in both mammalian and arthropod systems. The approach is to create virus-like particles with a fluourescent tag that can be used in various binding assays. The expression constructs have been developed and protocols to purify the VLP are being tested. Identification of genes essential for TBFV infections and antivirals. In the past year, our work on genome wide screens for host factors regulating TBFV infection has undergone analysis and should be ready for publication in the coming FY. Also in the past year, we began testing several libraries of small molecules against LGTV. Several promising compounds have been identified and will be evaluated to see if they might be useful antivirals. Development of an animal model for chronic POWV/DTV disease. Understanding how these viruses cause long-term symptoms as well as the possible role of viral persistence is important for developing therapies to treat chronic infection. However, there are not good animal models to study chronic disease. In the past year, we published our mouse model of chronic POWV/DTV disease. The results indicated that the continued presence of detectable virus infection was not required for chronic CNS disease, suggesting that host responses play an important role. In an interesting side project, we supplied a vial of 1940's vintage rocky mountain spotted fever vaccine, prepared from infected ticks, to Dr Taubenberger and his colleagues. Deep sequencing of the material revealed the the presence of Rickettsia ricketsii, demonstrating that that agent against which the vaccine designed was present. In addition, however, sequence reads of Coxiella burnetii, the agent of Q fever, were also identified. This likely indicated that some of the ticks were co-infected with both agents.

我们实验室的研究采用病毒学、免疫学、昆虫学、先进成像技术、基因组学、蛋白质组学、细胞生物学、分子生物学和载体生物学。我们在 BSL-2 中研究 LGTV，在 BSL-3 中研究 POWV/DTV。 I. scapularis 中的 TBFV 生物学。蜱虫感染是 TBFV 生物学的一个重要但尚未得到充分研究的特征。我们的离体器官培养工作已经获得了有关成年蜱中肠和唾液腺 TBFV 感染的大量信息，目前正在审查发表。去年，Ochwoto博士和Stewart博士采用人工膜血饲法，证明成年肩胛蜱可以通过喂食LGTV感染的血液而获得较高的感染效率。对进食成人中肠的研究揭示了肠上皮预期的分化和增殖为成熟的消化细胞，结果表明这些细胞支持病毒复制。初步研究表明，蜱虫进食时唾液腺也会被感染，并且唾液腺感染不需要预先感染中肠和随后的病毒全身传播。蜱虫的若虫阶段对于人类感染最为重要，因此去年我们开始研究若虫的感染情况。然而，当尝试使用人工膜系统感染它们时，结果令人失望且不一致。因此，Weck 博士计划通过喂食 LGTV 感染的小鼠来感染若虫。唾液腺至少有 3 种腺泡，每种腺泡含有多种细胞类型。目前尚不清楚是否所有腺泡类型或所有细胞类型都允许病毒感染。解决这个有趣问题的一种方法是感染离体唾液腺培养物，解离受感染的培养物并对细胞进行单细胞分析。去年，Ochwoto 博士和 Theriault 女士应用细胞分离方法解剖了成年蜱的唾液腺。使用简单的酶消化，腺泡难以解离，我们尚未获得足以进行分析的单细胞悬浮液。肩胛硬蜱中的微生物相互作用。肩胛硬蜱携带多种传染源，包括TBFV、伯氏疏螺旋体和嗜吞噬细胞无形体，但多种微生物的人类感染很少见。一种可能的解释是，一种微生物的感染会干扰下一秒的感染。去年，斯图尔特博士与弗吉尼亚理工大学的伊斯特伍德博士建立了合作，研究无形体和 TBFV 的双重感染。 Stewart 博士还在开发工具来识别哺乳动物和节肢动物系统中的 TBFV 受体。该方法是创建带有荧光标签的病毒样颗粒，可用于各种结合测定。表达构建体已经开发出来，纯化 VLP 的方案正在测试中。鉴定 TBFV 感染和抗病毒药物必需的基因。在过去的一年里，我们对调节 TBFV 感染的宿主因子进行全基因组筛选的工作已经过分析，应该可以在下一个财年发表。同样在去年，我们开始针对 LGTV 测试几个小分子库。几种有前景的化合物已经被鉴定出来，并将对其进行评估，看看它们是否可能是有用的抗病毒药物。慢性 POWV/DTV 疾病动物模型的开发。了解这些病毒如何引起长期症状以及病毒持续存在的可能作用对于开发治疗慢性感染的疗法非常重要。然而，目前还没有很好的动物模型来研究慢性病。去年，我们发表了慢性 POWV/DTV 疾病的小鼠模型。结果表明，慢性中枢神经系统疾病不需要持续存在可检测到的病毒感染，这表明宿主反应发挥着重要作用。在一个有趣的副项目中，我们向 Taubenberger 博士和他的同事提供了一瓶 1940 年老式落基山斑疹热疫苗，该疫苗是用受感染的蜱虫制备的。对材料的深度测序揭示了立克次氏体的存在，证明了疫苗设计所针对的病原体的存在。然而，此外，还鉴定了 Q 热病原体伯内氏柯克斯体的序列读数。这可能表明一些蜱虫同时感染了这两种病原体。

项目成果

期刊论文数量（32）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

DOI：
10.1371/journal.pone.0047912
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Offerdahl DK;Dorward DW;Hansen BT;Bloom ME
通讯作者：
Bloom ME

Genetic sequencing of a 1944 Rocky Mountain spotted fever vaccine.

DOI：
10.1038/s41598-023-31894-0
发表时间：
2023-03-22
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Xiao, Yongli;Beare, Paul A.;Best, Sonja M.;Morens, David M.;Bloom, Marshall E.;Taubenberger, Jeffery K.
通讯作者：
Taubenberger, Jeffery K.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

DOI：
10.1016/j.virol.2016.11.002
发表时间：
2017-01-15
期刊：
VIROLOGY
影响因子：
3.7
作者：
Offerdahl, Danielle K.;Dorward, David W.;Hansen, Bryan T.;Bloom, Marshall E.
通讯作者：
Bloom, Marshall E.

Characterization of flavivirus infection in salivary gland cultures from male Ixodes scapularis ticks.

DOI：
10.1371/journal.pntd.0008683
发表时间：
2020-10
期刊：
PLoS neglected tropical diseases
影响因子：
3.8
作者：
Kendall BL;Grabowski JM;Rosenke R;Pulliam M;Long DR;Scott DP;Offerdahl DK;Bloom ME
通讯作者：
Bloom ME

Modern dosimetric tools for (60)Co irradiation at high containment laboratories.

高密闭实验室中用于 (60)Co 辐照的现代剂量测定工具。