| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), existing in only the budded (enveloped) form, and Spodoptera frugiperda (Sf-9, Sf-21) and Trichoplusia ni BTI-TN-5B1-4 (High 5) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-^3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to insect cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before the cells were contacted with the mixture to extract the virus. The insect cells were then washed with a phosphate buffered saline, and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10^3 plaque forming unit per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses. The ELVA was also valuable to examine the stability of AcMNPV under various pH and temperature conditions.
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