MODULATION OF BOVINE LEUKEMIA VIRUS REPLICATION BY ANTIVIRAL DRUGS

抗病毒药物对牛白血病病毒复制的调节

• 批准号: 8360013
• 负责人: JEFFRY S ISAACSON
• 金额: $ 3.83万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360013
• 关键词: Animals; Antibody Formation; Antiviral Agents; B-Lymphocytes; BLV Infection; Biological Assay; Blood; Bovine Leukemia Virus; Cattle; Cell Line; Cells; Chronology; Development; Enhancers; Enhancing Antibodies; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Funding; Genetic; Giant Cells; Grant; Human Virus; Immune; Immunization; In Vitro; Infection; Life; Messenger RNA; Methods; Modeling; National Center for Research Resources; Nebraska; Peripheral Blood Mononuclear Cell; Principal Investigator; Proteins; Publishing; Reagent; Relative (related person); Reporting; Research; Research Infrastructure; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Source; Staging; Testing; Time; Titrations; United States National Institutes of Health; Viral Load result; Virus; Virus Diseases; Virus Replication; Work; cost; cytokine; functional genomics; immune activation; in vitro Model; inhibitor/antagonist; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 1. Optimize a BLV syncytium-forming assay for virus titration, and develop additional methods to study expression of BLV mRNA and proteins. Most published studies comparing various stages of BLV infection use peripheral blood mononuclear cells (PBMCs) isolated from the blood of naturally or experimentally infected cattle. The use of an in vitro model of BLV infection using various cell lines will allow better control of variables inherent in studying live animals (duration of infection, viral load, concomitant infections). In addition, being able to infect cells at a single time point will allow determination of the chronology of various effects in cells following viral infection. We are also optimizing a reverse-transcriptase PCR (RT-PCR) assay to study levels of various BLV RNAs in vitro, and we will also use ELISA and flow cytometry to study expression of BLV-encoded proteins. 2. Test various substances as potential modulators (inhibitors or enhancers) of BLV replication. No satisfactory treatment is available for HTLV, which infects about 20 million people worldwide (Gillet et al., 2007). Since BLV is a close genetic relative of HTLV, BLV should be a good model for testing potential inhibitors of HTLV, while avoiding the inherent risks of working with the human virus. Additionally, other compounds can be screened to determine effects on BLV replication (inhibition or enhancement), for use in further studies of BLV on host cells. 3. Use the compounds identified as inhibitor or enhancers of BLV replication in vitro as tools for studying of BLV effects on host immune cells. Prior to our development of the BLV-induced syncytia assay, we were mainly focused on investigating the mechanism(s) of immune activation by BLV. It has long been observed that peripheral blood mononuclear cells from BLV-infected animals undergo spontaneous proliferation in vitro (Trueblood et al., 1998). Moreover, the occurrence of elevated numbers of circulating B cells in about 30% of infected animals suggests that BLV may have an immunostimulatory effect. Additionally, several investigators have reported enhanced antibody responses (Isaacson et al., 1996a), increased B cell expression of MHC-II molecules (Isaacson et al., 1996b), and several alterations in cytokine secretion (Stone et al., 1994, Trueblood et al., 1998) associated with BLV infection. In the later part of this project, we plan to use any reagents identified as reliable inhibitors or enhancers of BLV replication in vitro to further elucidate the mechanism(s) of BLV-induced immunostimulation.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 1. 优化用于病毒滴定的 BLV 合胞体形成测定，并开发其他方法来研究 BLV mRNA 和蛋白质的表达。 大多数已发表的比较 BLV 感染各个阶段的研究都使用从自然或实验感染牛的血液中分离的外周血单核细胞 (PBMC)。 使用各种细胞系的 BLV 感染体外模型将可以更好地控制研究活体动物所固有的变量（感染持续时间、病毒载量、伴随感染）。 此外，能够在单个时间点感染细胞将允许确定病毒感染后细胞中各种影响的时间顺序。 我们还在优化逆转录酶 PCR (RT-PCR) 测定，以在体外研究各种 BLV RNA 的水平，并且我们还将使用 ELISA 和流式细胞术来研究 BLV 编码蛋白的表达。 2. 测试各种物质作为 BLV 复制的潜在调节剂（抑制剂或增强剂）。 HTLV 尚无令人满意的治疗方法，全世界约有 2000 万人感染该病毒（Gillet 等人，2007 年）。 由于 BLV 是 HTLV 的近亲，BLV 应该是测试 HTLV 潜在抑制剂的良好模型，同时避免使用人类病毒的固有风险。 此外，还可以筛选其他化合物以确定对 BLV 复制的影响（抑制或增强），用于进一步研究 BLV 对宿主细胞的作用。 3. 使用被鉴定为体外 BLV 复制抑制剂或增强剂的化合物作为研究 BLV 对宿主免疫细胞影响的工具。 在开发 BLV 诱导的合胞体测定之前，我们主要致力于研究 BLV 免疫激活的机制。 长期以来，人们观察到感染 BLV 的动物的外周血单核细胞在体外会发生自发增殖（Trueblood 等，1998）。 此外，约 30% 的受感染动物中循环 B 细胞数量增加表明 BLV 可能具有免疫刺激作用。 此外，一些研究人员报告了与 BLV 感染相关的抗体反应增强（Isaacson 等人，1996a）、MHC-II 分子 B 细胞表达增加（Isaacson 等人，1996b）以及细胞因子分泌的一些改变（Stone 等人，1994；Trueblood 等人，1998）。 在该项目的后期部分，我们计划使用任何被鉴定为可靠的 BLV 体外复制抑制剂或增强剂的试剂来进一步阐明 BLV 诱导的免疫刺激机制。