| Background Dengue is now the most rapidly spreading mosquito-borne viral disease in the world. In the last50years, incidence has increased30-fold with increasing geographic expansion to new countries. It has rapidly invaded the United States and Europe in the past two decades and has become a significant public health concern and, in the present decade, from urban to rural settings. An estimated50million dengue infections occur annually and approximately2.5billion people live in dengue endemic countries. In mainland China, dengue epidemics have spread from the southern coastal regions to the relatively northern and western regions since the1990s.Dengue is caused by dengue viruses (DENV) that belong to the genus Flavivirus of the family Flaviviridae. DENV is the most important mosquito-borne virus. It is an enveloped, single-stranded, positive-sense RNA virus (+ssRNA) with a genome size of approximately11,000nucleotides. The genome of DENV contains a single open reading frame (ORF) flanked by conserved5’ and3’ untranslated region (UTR). The ORF encodes a polyprotein precursor of approximately3400amino acids in length. This polypeptide is co-and posttranslationally processed give rise to three structural and seven nonstructural (NS) proteins, the structural proteins core (C), premembrane (prM), and envelope (E) and the NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). DENV is comprised of four antigenically distinct serotypes (DENV-1, DENV-2, DENV-3and DENV-4), multiple genotypes enclosed in each serotype. The mosquito vectors, principally Aedes aegypti (Ae. aegypti), and the second Aedes albopitus (Ae. albopitus)become infected after feeding on a viremic individual, then a reported the extrinsic incubation period (EIP) of7-14days is required before the mosquitoes can transmit the virus to a new host. DENV present in the blood meal firstly invades the midgut, then it encounters and must overcome midgut infection and escape barriers, and eventually disseminates throughout the mosquito, which becomes infectious once virus reaches the salivary glands, at which point the mosquito is infectious and has thus completed the EIP. In China mainland, Ae. albopitus is the primary mosquito vector of DENV. Mosquito bites after EIP result in human infection. Symptomatic dengue virus infections were grouped into three categories:undifferentiated fever, dengue fever (DF) and dengue haemorrhagic fever (DHF). DHF was further classified into four severity grades, with grades III and IV being defined as dengue shock syndrome (DSS).Although the global burden of DENV represents a growing challenge to public health, no specific therapeutics or licensed vaccines against DENV are available yet. There is no better alternative than vector control to reduce or prevent dengue virus transmission currently.MicroRNA (miRNA) has become quite a hot area of study right now. It was first identified in Caenorhabditis elegans in1998, since this initial observation,>30000mature miRNA products have been identified in206species according to the miRBase (version20.0). MiRNAs are an abundant group of endogenous non-protein coding small RNAs of18-25nucleotides that post-transcriptionally regulate gene expression. In insect, miRNA biogenesis begins with the nuclear transcription of long primary miRNAs (pri-miRNAs) by RNA polymerase II. The primary miRNA (pri-miRNA) are processed by the Drosha-Pasha complex to form a～70nt precursor miRNA (pre-miRNA) in the nucleus. Following export to the cytosol, the pre-miRNA is recognized by Dicer to form the miRNA:miRNA*duplex. One strand of the miRNA:miRNA*duplex is loaded into the RNA-induced silencing complex (RISC)and guided to mRNAs, resulting in either translational inhibition or mRNA degradation through perfect or partial complementarity between miRNAs seed region and the mRNAs targets.MiRNAs have been reported to play an important role in the regulation of embryonic development, stem cell differentiation, apoptosis, tumorigenesis and immunity. Currently, miRNAs were suggested to involve in virus-host interaction. Recent research suggests that host cell may use its own miRNA to regulate viral infection. Typically regulation is cellular miRNAs may repress virus replication. Nathans suggested that a highly abundant miRNA in HIV-1-infected human T lymphocytes, miR-29a, may suppress HIV-1production and infectivity. It was also reported that cellular miR-32acted as an antiviral defense against the retrovirus primate foamy virus type1(PFV-1). But on the other hand, cellular miRNAs may accelerate virus replication. It is reported that the interaction between the5’-UTR of the HCV and miR-122is indispensable for HCV replication in cultured cells.Recently, mosquito miRNAs have also been reported to positively or negatively modulate the host response to pathogen infection. Two miRNAs of the mosquito Culex quinquefasciatus(C. quinquefasciatus) showed significant changes in expression levels following WNV infection, implying potential roles for mosquito miRNAs in viral infection. The knock-down of two important proteins in miRNA biogenesis, Dicerl and Argonautel, led to increased sensitivity to Plasmodium infection in Anopheles gambiae, supporting an involvement of miRNAs in the defense reaction. However, relatively little is known about the interactions between mosquito miRNAs and dengue virus.Objective Several miRNAs have recently being identified in Asian tiger mosquito, Ae. albopitus and it was found that some miRNAs are differentially expressed when infected with DENV-2. Base on these results, we further analysis the role of these miRNAs function in regulating DENV replication. In mosquitoes, midgut tissue plays important roles in DENV infection. It is the first barrier for pathogen transmission. In this study, we focus on miRNA that is specific expressed in such important tissue, mosquito midgut. DENV-2infectious model in mosquito Ae. albopictus and C6/36cell line were firstly constructed. Subsequently artificial miRNA mimics and coresponsed miRNA inhibitor, miRNA antagomiRs were synthesis to functional analysis the role miR-281in modulating DENV-2repliction both in vitro and in vivo. Currently, no specific therapeutics or licensed vaccines against DENV are yet available. There is no better alternative than vector control to reduce or prevent dengue virus transmission. Thus, understanding the viral and host determinants governing DENV replication can provide novel targets for mosquito vector control strategies.Methods1. The New Guinea C strain (NGC) of DENV-2was propagated in C6/36cells. For oral infection of mosquitoes throughout the study,3-to5-day-old female mosquitoes were infected through infectious blood meals. The media of uninfected C6/36cells were cultured under similar conditions and served as mock-infected controls.2. The expression profiles of miR-281in body parts of Ae. albopictus were confirmed using total RNA extracted from different body parts of female mosquitos: the head, thorax, midgut and leftover. Northern blot and RT-qPCR were used to analysis the expression levels of miR-281.3. The influence of miR-281on DENV-2viral replication was explored in the mosquito cell line C6/36. Cells were first transfected with mimics to achieve higher expression levels of miR-281or antagomiRs to deplete the endogenous miR-281. The relative expression of miR-281after overexpression and knock-down was evaluated by RT-qPCR at24hrs post-transfection. Subsequently, cells were infected with DENV-2, and the DENV-2abundance was determined by RT-qPCR at48hrs post infection.Western blot analysis was also performed using a DENV-2E protein-specific polyclonal antibody. The DENV-2titers in the supernatant of C6/36cells were further tested to investigate whether miR-281affected DENV-2infectivity.4. We used the antagomiR approach to deplete miR-281expression in vivo. Female mosquitoes were injected with antagomiR-281and control antagomiRs. Efficient knock-down of miR-281was confirmed by qRT-PCR analysis in both mosquitoes and midguts.To further confirm the effect of miR-281knock-down on viral replication in vivo, the injected female mosquitoes were infected with DENV-2at48hrs post-injection. Samples were collected and analyzed by RT-qPCR.5. To explore whether miR-281has a potential interaction with the DENV-2genome, potential binding sites of miR-281in the DENV-2genome were predicted using RNAhybrid. We also searched for a potential target for miR-281in the Ae. albopictus transciptome using RNAhybrid&miRanda.6. To investigate the interaction between the seed region of miR-281and the predicted biding site, we used an EGFP plasmid-based reporter system in the C6/36cell line. The DENV5’-UTR and the mutant5’-UTR sequence were cloned upstream of the EGFP gene in the pIB expression vector, resulting in the constructs pIB/5UTR-EGFP (target-5UTR) and pIB/mut-5UTR-EGFP (mut-5UTR), respectively. The constructs were co-transfected with miR-281or control mimics into C6/36cells. EGFP transcript levels were detected using RT-qPCR.Results1. DENV-2infectious model in Ae. albopictus and C6/36cells were constructed.2. MiR-281is specifically expressed in the Ae. albopictus midgut and is regulated by DENV-2infection both in mosquitoes and miguts at4dpi and7dpi. Increase was also found in C6/36cells at96h after DENV-2infection.3. Functional interventional studies of miR-281demonstrated that overexpression enhanced DENV-2replication and down-regulation suppressed DENV-2replication in C6/36cells.4. Consistent with the in vitro result, depletion of miR-281led to a decrease in DENV-2gRNA in vivo.5. We searched for a potential target for miR-281in the DENV-2genome using computational software. The predicted target of miR-281was located in the5’-UTR SLA structure of DENV-2(nt37-nt55), in a sequence that is conserved among different serotypes. We also searched for a potential target for miR-281in the Ae. albopictus transciptome,35and87potential targets were found using RNAhybrid and miRanda, respectivily.11potential targets were found both in RNAhybrid and miRanda analyses.6. Significantly higher EGFP transcript levels were detected in pIB/5UTR-EGFP and miR-281mimic co-transfected cells compared with those cells co-transfected with pIB/5UTR-EGFP and control mimics. Moreover, pIB/mut-5UTR-EGFP and miR-281co-transfected cells exhibited a significant decrease in EGFP expression compared with pIB/5UTR-EGFP and miR-281mimic co-transfected cells, while no significant difference was found betweenpIB/5UTR-EGFP and control mimic co-transfected cellsat48hrs post-transfection.ConclusionIn conclusion, miR-281has been found to be specifically expressed at a high level in the midgut of female adult mosquitoes and is up-regulated after DENV-2infection. Functional interventional studies confirm that miR-281positively regulates DENV-2replication. A target study indicates that there is an interaction between miR-281and the DENV-25’-UTR sequence in the context of the EGFP reporter. Further study will be important to confirm the interaction between miR-281and the5’-UTR in the context of the DENV genome and to identify the targets of miR-281in the mosquito. To our knowledge, this is the first report of a midgut-specific miRNA that is involved in DENV replication. |
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