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. 2019 Jan 18;12(1):44.
doi: 10.1186/s13071-018-3261-2.

Screening for differentially expressed miRNAs in Aedes albopictus (Diptera: Culicidae) exposed to DENV-2 and their effect on replication of DENV-2 in C6/36 cells

Jianxin Su  1   2 , Gang Wang  1   3 , Chunxiao Li  1 , Dan Xing  1 , Ting Yan  1 , Xiaojuan Zhu  1 , Qinmei Liu  1 , Qun Wu  1 , Xiaoxia Guo  4 , Tongyan Zhao  5
Affiliations

Screening for differentially expressed miRNAs in Aedes albopictus (Diptera: Culicidae) exposed to DENV-2 and their effect on replication of DENV-2 in C6/36 cells

Jianxin Su et al. Parasit Vectors. .

Abstract

Background: The mosquito Aedes albopictus is an important vector for dengue virus (DENV) transmission. The midgut is the first barrier to mosquito infection by DENV, and this barrier is a critical factor affecting the vector competence of the mosquito. However, the molecular mechanism of the interaction between midgut and virus is unknown.

Results: Six small libraries of Ae. albopictus midgut RNAs were constructed, three of which from mosquitoes that were infected with DENV-2 after feeding on infected blood, and another three that remained uninfected with DENV-2 after feeding on same batch of infected blood. A total of 46 differentially expressed miRNAs were identified of which 17 significant differentially expressed miRNAs were selected. Compared to microRNA expression profiles of mosquitoes that were uninfected with DENV-2, 15 microRNAs were upregulated and two were downregulated in mosquitoes that were infected with DENV-2. Among these differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-622 were verified by stem-loop qRT-PCR in samples from seven-day-infected and uninfected midguts and chosen for an in vitro transient transfection assay. miR-1767 and miR-276-3p enhanced dengue virus replication in C6/36 cells, and miR-4448 reduced dengue virus replication.

Conclusions: To our knowledge, this study is the first to reveal differences in expression levels between mosquitoes infected and uninfected with DENV-2 after feeding on an infected blood meal. It provides useful information on microRNAs expressed in the midgut of Aedes albopictus after exposure to the virus.

Keywords: Aedes albopictus; Dengue virus (DENV); Midgut; microRNA (miRNA).

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Conflict of interest statement

Ethics approval

All of the experimental protocols involving animals were approved by the Laboratory Animal Center of the State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Institutional Animal Care and Use Committee (IACUC, permit number BIME 2011-2009). The study of animals was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
a Scatter plots and fold changes comparing the midguts of infected and uninfected Ae. albopictus at different time points. 5B/5A is the (5-day uninfected midguts)/(5-day infected midguts after exposure to DENV-2), 7B/7A is the (7-day uninfected midguts)/(7-day infected midguts), etc. b Screened significantly differentially expressed miRNAs between the midguts of infected and uninfected Ae. albopictus at different time points after a DENV-2-infected blood meal
Fig. 2
Fig. 2
Verification of differentially expressed miRNAs by stem loop qRT-PCR. a Amplification curves from stem loop qRT-PCR. b Relative expression of miRNA by stem loop qRT-PCR
Fig. 3
Fig. 3
Relative expression of the four miRNAs in C6/36 by stem loop qRT-PCR and in the midguts of Ae. albopictus by high throughput sequencing
Fig. 4
Fig. 4
Relative expression of miRNAs in C6/36 cells at 24 and 72 h post-transfection by qRT-PCR
Fig. 5
Fig. 5
Relative expression of DENV-2 in C6/36 cells at 72 h post-transfection by qRT-PCR
Fig. 6
Fig. 6
Survival rates of C6/36 cells at 72 h post-infection with DENV-2 by MTT
See this image and copyright information in PMC

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