| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV), and considerable number of cattle have been shown to be infected with this virus; therefore, it is critical to clarify if human being can also be infected with this virus.
(1) Detection of BLV provirus DNA (pDNA)
BLV pDNA was amplified from peripheral blood mononuclear cells (PBMC) DNA with PCR and confirmed with Southern blotting (SB), or quantitated with real-time PCR. Although barely detectable BLV pDNA was found in a few samples, results of confirmatory experiments were inconsistent. Considering that proviral load might be around threshold level, we expanded peripheral blood B cells that are supposed to be BLV-targeting cells by transforming them with Epstein-Barr virus (EBV). Using large amount of DNA samples of EBV-transformed B cells derived from a healthy volunteer, we could amplified and sequenced several BLV genes such as tax, env and pol. Phylogenetic analysis of BLV env gene demonstrated the
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identified BLV pDNA was close to those in the USA and Australia and almost identical to those found in cows in Japan. However, amplification of more extended regions of BLV pDNA has been unsuccessful so far, partly because of difficulty in PCR and low copy number.
(2) Detection of anti-BLV antibody
BLV proteins were prepared from lysates of persistently BLV-infected cells or from cell-free culture supernatants of those cells. In addition, recombinant BLV Tax or Env protein was purified as fusion proteins with GST from E. coli cultures. Those BLV proteins were used as antigens for Western blotting (WB) to detect anti-BLV antibodies in human serum samples. Based on the diagnostic criteria for HTLV WB, one (9%) was positive and another (9%) was ambiguous among 11 healthy volunteers, and one (3%) was positive and three (9%) were ambiguous among 32 patients with breast cancer. However, the presence of BLV pDNA was not confirmed in BLV-seropositive individuals.
Discussion: Different from a recently reported study in the USA, there were not many BLV-seropositive individuals and the presence of anti-BLV antibody did not predict the presence of BLV pDNA in their peripheral blood B cells. Thus, BLV infection is probably rare, if any, in humans. Less
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