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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 1。优化对病毒滴定的BLV合成测定法，并开发出研究BLV mRNA和蛋白质表达的其他方法。 大多数已发表的研究比较了BLV感染的各个阶段使用外周血单核细胞（PBMC），这些细胞（PBMC）从自然或经过实验感染的牛的血液中分离出来。 使用各种细胞系的BLV感染的体外模型将可以更好地控制研究活体动物（感染持续时间，病毒载荷，伴随感染）固有的变量。 另外，能够在一个时间点感染细胞将允许在病毒感染后测定各种影响的年代学。 我们还将优化反向转录酶PCR（RT-PCR）测定法以研究各种BLV RNA的体外水平，我们还将使用ELISA和流式细胞仪研究BLV编码的蛋白质的表达。 2。测试各种物质作为BLV复制的潜在调节剂（抑制剂或增强子）。 HTLV无法获得令人满意的治疗方法，HTLV在全球范围内感染了约2000万人（Gillet等，2007）。 由于BLV是HTLV的亲密遗传亲戚，因此BLV应该是测试HTLV潜在抑制剂的好模型，同时避免使用人类病毒的固有风险。 此外，可以筛选其他化合物，以确定对BLV复制（抑制或增强）的影响，以在宿主细胞上进一步研究BLV。 3。使用在体外鉴定为抑制剂或BLV复制增强子的化合物作为研究BLV对宿主免疫细胞影响的工具。 在我们开发BLV诱导的合胞菌测定法之前，我们主要专注于研究BLV免疫激活的机制。 长期以来，已经观察到，来自BLV感染动物的外周血单核细胞在体外会自发增殖（Trueblood等，1998）。 此外，大约30％的感染动物中循环B细胞数量升高的发生表明BLV可能具有免疫刺激作用。 此外，一些研究者报道了抗体反应增强（Isaacson等，1996a），增加了MHC-II分子的B细胞表达（Isaacson等人，1996b），以及细胞因子分泌的几种改变（Stone等，1994，Trueblood等，1998，1998，1998）。 在该项目的后期，我们计划使用在体外鉴定为BLV复制的可靠抑制剂或增强子的任何试剂，以进一步阐明BLV诱导的免疫刺激的机制。