2014 Fiscal Year Research-status Report
Determination of the role of Fcgamma receptor in antibody-dependent enhancement of dengue virus infection
| Project/Area Number |
26870872
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| Research Institution | Nagasaki University |
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Principal Investigator |
モイ メンリン 長崎大学, 熱帯医学研究所, 准教授 (40597499)
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| Project Period (FY) |
2014-04-01 – 2017-03-31
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| Keywords | dengue / ADE / dengue hemorrhagic fever |
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| Outline of Annual Research Achievements |
To study the role of FcγR in infection-enhancement (ADE) of dengue virus (DENV) during infection, we focused on determination of the types of FcγR and receptor cytoplasmic regions which are involved in ADE. In FYI 2014, using molecular techniques, FcγRI, γ-chain and FcγRIIA genes were constructed and sub-clonned into mammalian expression vectors with antibiotic resistant cassettes to construct the FcγRIIA-pcDNA3.1(neo), FcγRIA-pCMV6A(bsd), and γ-chain-pCMV6A(puro) plasmids. The plasmids were then transfected into a baby hamster kidney cell (BHK) cell line using transfection reagents, and cell surface expression of FcγRIIA and FcγRIA, and expression of γ-chain in the intercellular compartment in each of the cell lines were confirmed using flow-cytometry and western blot. Because FcγRIA requires an activation chain (γ-chain) to function, dual transfection of FcγRIA-pCMV6A(bsd) and γ-chain-pCMV6A(puro) plasmids into BHK cells was also performed. Antibiotic-resistant cells were then selected and were further selected for resistant colonies using the limited-dilution method. Resistant colonies were then selected using protein expression as a criteria by flow-cytometry. Four cell lines stably expressing the FcγRIA, FcγRIIA, γ-chain and the FcγRIA-γ chain were successfully established and propagated.
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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
FcγR-expressing plasmids were constructed and protein expression of each of the receptors were confirmed. Cell lines stably expressing the receptors were successfully generated. The cell lines would prove useful for further studies on ADE in DENV infection.
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| Strategy for Future Research Activity |
Constructs of the FcγR with altered cytoplasmic regions would be generated and the role of each receptor in ADE will be determined using these cell lines by DENV infection assay. In addition, using the stable cell lines developed in the previous year, cellular factors and markers involved in ADE antibody immune-complex internalization via the FcγR will be determined, to establish fundamental knowledge and novel approaches for DENV therapeutics and clinical applications.
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| Causes of Carryover |
(1) Travel expenses planned for research presentation were partially covered by another funding.
(2) Lesser consumables were purchased for gene construction and transfection because the experiments went well.
(3) Due to importation/manufacturer issues, purchases of consumables (reagents such as antibodies for receptor detection) planned for the previous year will be carried forward to the next financial year.
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| Expenditure Plan for Carryover Budget |
Incurring amount will be used for:
1. Tools and consumables for cell culture and molecular (protein and genomic) studies. 2. Travel expenses for participation and presentation in local and international meetings and conferences, to (a) disseminate research achievements to the general public, (b) promote information exchange, and (c) gather new information for further research advancement. 3. Publication fees and proofreading to publish findings and disseminate results to society and citizens.
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[Journal Article] Two cases of Zika fever imported from French Polynesia to Japan, December 2013 to January 2014.2014
Author(s)
Kutsuna S, Kato Y, Takasaki T, Moi ML, Kotaki A, Uemura H, Matono T, Fujiya Y, Mawatari M, Takeshita N, Hayakawa K, Kanagawa S, Ohmagari N.
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Journal Title
Euro Surveillence
Volume: 19(4)
Pages: pii20683
DOI
Peer Reviewed / Open Access
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[Presentation] Neutralizing antibody titers as a surrogate for protection against dengue: a revisit of neutralizing antibody titers of dengue virus using FcγR-expressing cells.2014
Author(s)
Moi ML, Rattanamahaphoom J, Lim CK, Sirivichayakul C, Saijo M, Sabchareon A, Takasaki T, Kurane I.
Organizer
Joint International Tropical Meeting (JITMM)
Place of Presentation
Centara Grand, Central World, Bangkok, Thailand
Year and Date
2014-12-02 – 2014-12-04
Invited
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[Presentation] Demonstration of common marmosets (Callithrix jacchus) as a non-human primate model for dengue vaccine development2014
Author(s)
Moi ML, Shirai K, Ami Y, Miyata Y, Lim CK, Suzaki Y, Kitaura K, Saijo M, Suzuki R, Kurane I, Takasaki T.
Organizer
第 62 日本ウイルス学会学術集会
Place of Presentation
神奈川県横浜市、パシフィコ横浜 会議センター
Year and Date
2014-11-10 – 2014-11-12
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[Presentation] Development of a non-human primate model for primary and secondary dengue virus infection using marmosets (Callithrix jacchus).2014
Author(s)
Moi ML, Shirai K, Ami Y, Lim CK, Suzaki Y, Kitaura K, Saijo M, Suzuki R, Takasaki T, Kurane I.
Organizer
The 63rd Annual Meeting of the American Society of Tropical Medicine and Hygiene
Place of Presentation
New Orleans Marriott, New Orleans, Lousiana, USA
Year and Date
2014-11-02 – 2014-11-06
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