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

在本财政年度，我们尝试通过向蚊子胸部注射l（2）efl-1、-2、-3和-4 dsRNA，然后在注射dsRNA后48小时注射DENV-2来体内分析基因。我们使用与在CCL-125中转染的相同的dsRNA。我们做了初步实验，看看dsRNA在体内会产生多长时间的效果。我们发现，注射dsRNA后72小时，l（2）efl-1至l（2）efl-5的水平受到抑制。因为我们需要考虑72小时后dsRNA可能失去其作用，所以我们决定在dsRNA注射后48小时注射DENV-2。注射dsRNA后，蚊子看起来很健康，只有少量蚊子死亡。48小时后，我们给蚊子注射DENV-2，并在24、48和72小时后处死蚊子。不幸的是，通过qRT-PCR，我们发现注射dsRNA的蚊子和对照蚊子之间没有显著差异，DENV-2继续复制的原因可能是在注射DENV-2时dsRNA失去了作用。在未来，我们将尝试同时共注射DENV-2和dsRNA，或者通过在胚胎阶段注射dsRNA来创建转基因蚊子。我们已将体外实验的结果提交给日本传染病杂志供考虑。

On this fiscal year, we tried to analyze the gene in-vivo by injecting l(2)efl-1, -2, -3, and -4 dsRNAs to mosquito thoraxes and then followed by DENV-2 injection 48 hours after dsRNA-injection. We used the same dsRNA that we transfected in CCL-125. We did preliminary experiment to see how long the dsRNA would give an effect in-vivo. We found that 72 hours after dsRNA injection, the level of l(2)efl-1 to l(2)efl-5 was suppressed. Because we needed to consider that after 72 hours dsRNA might be losing its effect, we decided to inject DENV-2 48 hours after dsRNA injection. Following dsRNA-injection, mosquitoes looked healthy with only little number of mosquitoes died. Forty eight hours afterwards, we injected mosquitoes with DENV-2 and sacrificed the mosquitoes 24, 48, and 72 hours later. Unfortunately, using qRT-PCR, we found that there was no significant difference between dsRNA-injected and control mosquitoes.The reason that DENV-2 kept on replicating might because dsRNA was losing its effect at the time of DENV-2 injection. In the future, we will try to co-inject DENV-2 and dsRNA at the same time or to create a transgenic mosquito by injecting dsRNA at the embryo stage. We have submitted the results of in-vitro experiment to Japanese Journal of Infectious Disease for consideration.