2015 Fiscal Year Research-status Report
Determination of the role of Fcgamma receptor in antibody-dependent enhancement of dengue virus infection
| Project/Area Number |
26870872
|
|---|
| Research Institution | Nagasaki University |
|---|
Principal Investigator |
モイ メンリン 長崎大学, 熱帯医学研究所, 准教授 (40597499)
|
|---|
| Project Period (FY) |
2014-04-01 – 2017-03-31
|
| Keywords | dengue / antibody |
|---|
| Outline of Annual Research Achievements |
The objective of this research is to define the role of Fcgamma receptor in infection-enhancement (ADE) of dengue virus (DENV). Here, we have constructed the plasmids of Fcgamma receptor I, IIA, III and gamma chain, and expressed the respective plasmids into BHK cells. The expression of the receptors were examined by flow-cytometry and the percentage of expression was >50% for each of the cell lines. Next, cell lines that stably express each of the receptors were selected under antibiotic selection.
Using these cell lines, DENV infectivity was examined using conventional plaque assay. However, plaque formation rates were significantly lower in comparison to BHK parent cell lines. The results suggest that the protein expression and stable cell line selection may have resulted in cells that less permissive to DENV infection in comparison to parent cell lines.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The research was slightly delayed due to maternity leave.
|
| Strategy for Future Research Activity |
The DENV infectivity wil be examined using cell lines transiently expressing the Fcgamma receptors and the results will be compared to those of stable cell lines. Additionally, the levels of virus genome in cell culture supernatant will be determined by using quantitative real-time PCR.
In addition, cellular factors involved in ADE of DENV infection via the FcgammaR will be determined by using RT-PCR array and quantified by digital PCR. Confirmation of the protein expression of these factors will be performed by using western blot.
The final aim of this study is to determine the cellular factors involved in ADE of DENV infection, and to establish novel approaches for DENV therapeutics and clinical applications.
|
| Causes of Carryover |
The research was delayed due to maternity leave and part of the planned experiments could not be conducted.
|
| Expenditure Plan for Carryover Budget |
The incurring amount will be used according to the research plan with the addition of a supporting study to determine cellular pathway activation during DENV infection, in the presence of infection-enhancement antibodies and in comparison with other flavivirus. Other than Fcgamma receptor expressing cells, human hematopoetic cell lines that possess the Fcgamma receptor will also be used for comparison. Additionally, the associated genome expression of downstream signal of Fcgamma receptor during DENV infection will be determined by using RT-PCR array and confirmed by protein detection. The funds will also be used to publish data generated from this research theme.
|
