MODULATION OF BOVINE LEUKEMIA VIRUS REPLICATION BY ANTIVIRAL DRUGS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 1.优化BLV合胞形成病毒滴定方法，建立研究BLV基因和蛋白表达的新方法。大多数已发表的比较BLV感染不同阶段的研究使用了从自然或实验感染的牛的血液中分离出来的外周血单核细胞(PBMCs)。使用不同细胞系的BLV感染体外模型的使用将允许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聚合酶(RT-PCR)方法，以研究BLV体外各种RNA的水平，我们还将使用ELISA法和流式细胞术来研究BLV编码蛋白的表达。 2.测试多种物质作为BLV复制的潜在调节剂(抑制剂或增强剂)。HTLV感染了全世界约2000万人，目前还没有令人满意的治疗方法(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，同时避免了与人类病毒合作的固有风险。此外，还可以筛选其他化合物来确定对BLV复制的影响(抑制或增强)，用于进一步研究BLV对宿主细胞的影响。 3.将体外鉴定为BLV复制抑制或促进因子的化合物作为研究BLV对宿主免疫细胞影响的工具。在我们发展BLV诱导的合胞实验之前，我们主要致力于研究BLV激活免疫的机制(S)。长期以来，人们观察到BLV感染动物的外周血单核细胞在体外经历了自发的增殖(Truehemed等人，1998)。此外，在大约30%的感染动物中，循环B细胞数量的增加表明BLV可能具有免疫刺激作用。此外，一些研究人员报告了与BLV感染有关的抗体反应增强(Isaacson等人，1996a)，MHC-II分子B细胞表达增加(Isaacson等人，1996b)，以及与BLV感染有关的细胞因子分泌的几个变化(Stone等人，1994，Trueath等人，1998)。在这个项目的后期，我们计划使用任何被确认为可靠的体外BLV复制抑制或促进的试剂来进一步阐明BLV诱导免疫刺激的机制(S)。

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.