State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, People's Republic of China.
Center for Disease Control and Prevention of Guangzhou Military Region, Guangzhou, 510507, People's Republic of China.
Parasit Vectors. 2017 Feb 3;10(1):63. doi: 10.1186/s13071-017-1966-2.
The midgut is the first barrier to dengue virus (DENV) infections of mosquitoes and therefore is a major bottleneck for the subsequent development of vector competence. However, the molecular mechanisms responsible for this barrier are unknown.
We constructed three small RNA libraries from the midguts of adult Aedes albopictus females that had been fed on either sugar solution, an uninfected blood meal, or a blood meal infected with DENV-2, and112 conserved microRNAs represented by 173 miRNA sequences were identified, with 34 novel microRNAs predicted by Mireap, RNAfold and Sfold software. In addition, the expression of aal-miR-1174, aal-miR-2951 and aal-miR-956 was confirmed via stem-loop quantitative real-time PCR (qRT-PCR). Compared with microRNA expression profiles of mosquitoes that had ingested a regular blood meal, 43 microRNAs were upregulated and 4were downregulated in mosquitoes that had ingested a DENV-2-infected blood meal. Among the differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-4728-5p were verified via stem-loop qRT-PCR.
Analyses indicated that the changing patterns in miRNA expression during DENV-2 infection were significant and varied at different time points post infection. Most miRNA were upregulated at 24 h but were downregulated at 48 h post DENV-2 intake. The aal-miR-4728-5p was chosen for an in vitro transient transfection assay, and the results show that this miRNA enhances DENV replication in C6/36 cells. This study provides the first information on microRNAs expressed in the midgut of Ae. albopictus and describes species-specific changes in their expression levels following infection by DENV-2.
中肠是登革病毒（DENV）感染蚊子的第一道屏障，因此是后续媒介效能发展的主要瓶颈。然而，负责这一屏障的分子机制尚不清楚。
我们从已吸食糖溶液、未感染的血餐或感染 DENV-2 的血餐的成年白纹伊蚊雌蚊的中肠构建了三个小 RNA 文库，鉴定出 112 个保守 miRNA，代表 173 个 miRNA 序列，其中 34 个新的 miRNA 由 Mireap、RNAfold 和 Sfold 软件预测。此外，通过茎环定量实时 PCR（qRT-PCR）证实了 aal-miR-1174、aal-miR-2951 和 aal-miR-956 的表达。与吸食常规血餐的蚊子的 miRNA 表达谱相比，吸食 DENV-2 感染血餐的蚊子中有 43 个 miRNA 上调，4 个 miRNA 下调。在差异表达的 microRNAs 中，通过茎环 qRT-PCR 验证了 miR-1767、miR-276-3p、miR-4448 和 miR-4728-5p。
分析表明，在 DENV-2 感染期间 miRNA 表达的变化模式显著，并且在感染后不同时间点有所不同。大多数 miRNA 在 24 小时时上调，但在 DENV-2 摄入后 48 小时时下调。选择 aal-miR-4728-5p 进行体外瞬时转染试验，结果表明该 miRNA 增强了 C6/36 细胞中 DENV 的复制。本研究首次提供了白纹伊蚊中肠表达的 microRNAs 信息，并描述了感染 DENV-2 后其表达水平的种特异性变化。
