MODULATION OF BOVINE LEUKEMIA VIRUS REPLICATION BY ANTIVIRAL DRUGS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 1. 优化用于病毒滴定的BLV合胞体形成试验，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感染不同阶段的研究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感染体外模型将允许更好地控制研究活动物中固有的变量（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效应的时序。 我们还优化了逆转录酶PCR（RT-PCR）检测，以研究各种BLV RNA在体外的水平，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）。 对于HTLV没有令人满意的治疗，其感染了全世界约2000万人（Gillet et al.，2007年）。 由于BLV是HTLV的近亲，因此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，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. 使用体外鉴定为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。 在我们开发BLV诱导的合胞体测定之前，我们主要集中于研究BLV免疫激活的机制。 长期以来观察到来自BLV感染动物的外周血单核细胞在体外经历自发增殖（Trueblood等人，1998年）。 此外，在约30%的感染动物中出现循环B细胞数量升高，表明BLV可能具有免疫刺激作用。 此外，一些研究者已经报道了增强的抗体应答（Isaacson et al.，1996 a），增加MHC-II分子的B细胞表达（Isaacson等，1996 b），以及细胞因子分泌的几种改变（Stone等，1994年，Trueblood等人，1998）与BLV感染有关。 在本项目的后期，我们计划使用任何被鉴定为BLV体外复制的可靠抑制剂或增强剂的试剂来进一步阐明BLV诱导的免疫刺激的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.