2018 Fiscal Year Annual Research Report
Investigation of bovine leukemia virus-induced leukemogenesis
| Project/Area Number |
18F18100
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
間 陽子 国立研究開発法人理化学研究所, 開拓研究本部, 研究員 (50182994)
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| Co-Investigator(Kenkyū-buntansha) |
MAIREPATI PALATI 国立研究開発法人理化学研究所, 開拓研究本部, 外国人特別研究員
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| Project Period (FY) |
2018-04-25 – 2020-03-31
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| Keywords | Bovine leukemia virus / human Blood / human cancer tissue / DNA extraction / PCR amplification / Next-generation sequencing / BLV-human hybrid genome / BLV integration site |
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| Outline of Annual Research Achievements |
Recently, a scientific controversy of whether the presence of BLV in human and of the possible association of BLV with human disease result in serious public health concerns worldwide and require further investigations to explore BLV as a risk factor for human health. For this aim, We obtained 100 human blood samples from Japanese red cross and 27 breast cancer tissue sample from school of medicine Nihon University. All 127 human samples were subjected to DNA extraction. To determine the presence of BLV in human, we used BLV provirus specific primers to amplify targeting proviral genome such as LTR, gag, pol, env and tax through PCR. Among 100 tested blood samples, 6 sample showed specific BLV-positive band, and 5 out of 27 best cancer tissues were screened as BLV positive. To further investigate the BLV integration sites in human genome, these BLV-positive samples were subjected to highthrough-put next-generation sequencing. NGS data were visualized by IGV software and screening of NGS data (Chromosome 9~22) of blood samples showed 889 possible candidate BLV integration sites which needs to be confirmed by PCR to get BLV-human Hybrid genome. Data analysis of the rest of NGS data of blood samples and of breast cancer samples might result in more possible candidate BLV integration sites.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Understanding better the association between BLV and human disease, DNA were extracted from 127 human samples including blood and tissue samples. The presence of BLV in human was firstly determined by the amplification of BLV structural and regulatory genes (LTR, gag, pol, env, and tax) using BLV-specific primers by PCR. Secondly, BLV positive human samples then were subjected to make DNA library for highly sensitive Miseq sequencing. The preliminary result of NGS data of human samples obtained from Miseq were visualized by IGV software, and up to now about 889 candidate BLV integration sites were determined. Now I am still analyzing the rest of NGS data and will perform PCR to confirm those candidate sites. In addition, I am currently optimizing experiment method for the next step.
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| Strategy for Future Research Activity |
The rest of the NGS data of blood samples and cancer tissue samples will be screened to find possible BLV integrated sites in human genome and candidate sites are further confirmed by PCR amplification of hybridized BLV-human genome. The possible factors affecting on distinctive BLV integration (if available) will be studied. If our NGS data shows clear BLV integration in human genome, then BLV infectivity in human would be investigated by Luminescence Syncytia Induction Assay (LuSIA). For Observing viral expression, infected cells would be cultured and detecting viral proteins by western blotting and immunofluorescent staining assays (IFA). Analyze and summarize all data to write manuscript about the presence and integration of BLV in human.
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