研究課題
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.
1: 当初の計画以上に進展している
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。
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).
すべて 2015 2014
すべて 雑誌論文 (2件) (うち査読あり 2件、 オープンアクセス 2件) 学会発表 (5件)
Urban Pest Managament
巻: 4 ページ: 1-14
Parasites & Vectors
巻: 7 ページ: 143
10.1186/1756-3305-7-143
