デングウイルス感染蚊の網羅的遺伝子発現と新規ベクタ－コントロール

2014 Fiscal Year Annual Research Report

• Project/Area Number: 14J12097
• Research Institution: Oita University
• Principal Investigator: ルントゥウェネ L.R. 大分大学, 医学部, 特別研究員(PD)
• Project Period (FY): 2014-04-25 – 2016-03-31
• Keywords: dengue virus / vector mosquito / RNA-seq / qRT-PCR / L(2)efl / siRNA / CCL-125 / Aedes aegypti

Outline of Annual Research Achievements

Using RNA-seq to compare messenger RNA (mRNA) abundance of dengue-infected yellow fever mosquitoes to non-infected, we found that many genes were regulated due to the infection. Because RNA-seq reliability was still unknown, we validated our RNA-seq data with a different method, which was qualitative RT-PCR (qRT-PCR). We were able to find a Spearman correlation coefficient of 0.85 (p = 0.001) between RNA-seq and qRT-PCR, which may be interpreted that the association between the two assays would be considered statistically significant.
We analyzed further one gene from our RNA-seq data: L(2)efl. L(2)efl expression was up-regulated when we infected yellow fever mosquito cells (CCL-125) with dengue virus type 2 (DENV-2) or with synthetic double-strand RNA (poly(I:C)). This up-regulation was also found in mosquito and the result was in concordance to RNA-seq experiment. We hypothesized that L(2)efl gene might help to reduce DENV-2 replication in mosquito. To further analyze it, we first stimulated L(2)efl expression by administering poly(I:C) to CCL-125 and subsequently infecting the pre-treated cells with DENV-2. In the pre-treated cells, we found that DENV-2 titer was lower than in control.
In the L(2)efl-suppressed cells, we found that DENV-2 titer was higher compared to control. Using immunoblotting, we could show that eIF2α was phosphorylated when cells were treated with DENV-2 or poly(I:C). Also, whether the same phenomenon happens in live mosquito is still unclear. We are planning to analyze the role of L(2)efl to restrict DENV-2 replication in-vivo.

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

当初、次世代シーケンサーを用いて、RNA-seqによる解析データのみを得る事を想定していたところ、標的遺伝子の一つを発見した。その興味ある標的遺伝子の免疫学的パスウエイを解析する必要に迫られた頃、免疫学者との強力な共同研究が可能となった。このことにより、標的遺伝子の免疫学的手法による解析を押し進める原動力となった。

Strategy for Future Research Activity

The next experiment will involve live mosquitoes to see the introduction of L(2)efl dsRNA will favor DENV-2 replication in vivo. We will use 1-4 days old yellow fever mosquito. Using microinjection, we will inject L(2)efl dsRNA to the mosquitoes' thorax. Within 72 hours, we will subsequently inject the mosquitoes with live DENV-2 and then check the viral genome in the mosquitoes after 24 and 48 hours and compared them to control mosquitoes (only random dsRNA).

Research Products

• [Journal Article] Vector competence of Aedes aegypti to dengue virus (2014)
  • Author(s): Lucky R. Runtuwene, Kaori Noguchi, Akinori Tokunaga, Takashi Kobayashi, Kenta Nakai, Yuki Eshita
  • Journal Title: Urban Pest Managament Volume: 4 Pages: 1-14
  • Peer Reviewed / Open Access
• [Journal Article] Dengue transmission model by means of viremic adult immuno-competent mouse (2014)
  • Author(s): Lucky Ronald Runtuwene, Eiji Konishi, Atsushi Yamanaka, Yoshihiro Makino, Yutaka Suzuki, Tomohiko Takasaki, Ichiro Kurane, Takashi Kobayashi and Yuki Eshita
  • Journal Title: Parasites & Vectors Volume: 7 Pages: 143
  • DOI: 10.1186/1756-3305-7-143
  • Peer Reviewed / Open Access
• [Presentation] 日本産Aedes (Stegomyia) scutellarisグループ蚊のチクングニアウイルス媒介能について (2015)
  • Author(s): 福田昌子、Lucky R. Runtuwene、野口香緒里、Thipruethai Phanitchat、Raweewan Srisawat、Narumon Komalamisra、牛島廣治、倉根一郎、高崎智彦、小林隆志、江下優樹
  • Organizer: 第67回日本衛生動物学会大会
  • Place of Presentation: 金沢大学宝町キャンパス（石川県金沢市）
  • Year and Date: 2015-03-27 – 2015-03-29
• [Presentation] Susceptibility of Japanese Aedes mosquitoes to arboviruses (2015)
  • Author(s): Yuki Eshita, Masako Fukuda, Lucky R. Runtuwene, Shinya Hidano, Kyoko Hayashida, Raweewan Srisawat, Narumon Komalamisra, TomohikoTakasaki, Ichiro Kurane, Hironari Narita, Hiroshi Ushijima, Junya Yamagishi, Chihiro Sugimoto, Yutaka Suzuki and Takashi Kobayashi
  • Organizer: Second International Workshop on Aedes albopictus, the dengue vector
  • Place of Presentation: Guanzou, P.R. China
  • Year and Date: 2015-03-23 – 2015-03-25
• [Presentation] RT-LAMP法の改良によるアルボウイルス症診断の検討 (2014)
  • Author(s): 岩中 愛、松原祥恵、福田昌子、Thipruethai Phanitchat、Raweewan Srisawat、Lucky R. Runtuwene、野口香緒里、Arthur E. Mongan、鈴木 穣、Narumon Komalamisra、成田弘成、森田公一、倉根一郎、 高崎智彦、林田京子、杉本千尋、小林隆志、江下優樹
  • Organizer: 第26回日本環境動物昆虫学会年次大会
  • Place of Presentation: 長崎大学大学教育学部（長崎県長崎市）
  • Year and Date: 2014-11-29 – 2014-11-30
• [Presentation] Potential novel anti-dengue in Aedes aegypti mosquito (2014)
  • Author(s): Lucky R. Runtuwene, Shuichi Kawashima, Kaori Noguchi, Yutaka Suzuki, Sumio Sugano, Kenta Nakai, Ryuichiro Maeda, Junya Yamagishi, Chihiro Sugimoto, Tomohiko Takasaki, Ichiro Kurane, Yuki Eshita and Takashi Kobayashi
  • Organizer: 第37回日本分子生物学会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] 一過性的にデングウイルス血症をマウスに起こして、定量的感染蚊を大量に作る新規の方法 (2014)
  • Author(s): 江下優樹, Lucky R.Runtuwene, 小西英二, 山中敦史, 牧野芳大,野口香緒里, 川上絵理, 福田昌子, 小林隆志, Raweewan Srisawat, Narumon Komalamisra, 成田弘成, 牛島廣治, Arthur E. Mongan, 今田美穂子,山岸潤也, 鈴木 穣, 中井謙太, 前田龍一郎, 杉本千尋, 倉根一郎, 高崎智彦
  • Organizer: 第35回都市有害生物管理学会大会・総会
  • Place of Presentation: 日本大学生産工学部（千葉県県習志野市）
  • Year and Date: 2014-06-28 – 2014-06-29