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.
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