Identification And Screening Of MicroRNAs Related With Dengue Virus Infection Specifically Expressed In The Midgut Of Ae.Albopictus

Dengue fever (DF) and Dengue hemorrhagic fever (DHF) are the most seriousvector-borne diseases which are widely spreaded in tropical and sub-tropical region.Aedes aegypti L. and Ae. albopictus Skuse are the principal vectors of DENV. Becausea dengue fever vaccine has yet to be developed, vector control is currently the onlyeffective means of preventing this disease. However, despite the efforts on vectorcontrol, the global pandemic of DF has increased dramatically in recent decades, and,therefore, alternative control strategies are being investigated. Some of these effortshave focused on the genetic manipulation of insect vectors to modulate characteristicssuch as vector competence, but manipulation of vector competence must based onextensive knowledge on the molecular factors of vector-pathogen interactions.The ability of vectors to infect a pathogen is a key index of vector competence andseveral barriers hinder the infection of mosquito vectors by arboviruses. Of these, themidgut is the first barrier to the invasion of pathogens ingested into the alimentary tractand the ability of midgut epithelial cells to resist viral infection is the main factordetermining the susceptibility of mosquitoes to arbovirus infection. Howerer, themolecular mechanisms underlying the specific binding between viral pathogens andmidgut epithelial cells that regulate viral replication are still unclear. This in turn hasbeen an obstacle to research on the molecular mechanisms responsible for thesusceptibility of mosquito vectors to DENV. Understanding the molecular mechanismsunderlying these differences in vector competence is important to assessing the riskposed by any particular arbovirus and mosquito vector combination and developingnovel strategies to mitigate or block the transmission of mosquito-borne arboviruses.MicroRNAs (miRNA), a novel class of gene regulators, are a class of non-codingRNA molecules that regulate gene expression at the post-transcriptional level. Severalstudies have shown that miRNA play important roles in controlling viral infection andconferring innate immunity. In Anopheles gambiae, knocking down Dicer1and Ago1 led to an increased sensitivity to Plasmodium infection in the midgut; in Aedes egypti, atransgenic family expressed inverted-repeat RNA in the midgut was constructed andthese mosquitoes ingesting an artificial bloodmeal containing DENV-2exhibitedmarked reduction of viral replication in the midguts after infection. These experimentsprove that miRNA play important roles in midgut infection by the DENV. At least88miRNA have been identified in Ae. egypti(miRBase20) and several studies have shownthat DENV infection causes changes in the expression of miRNA in the midgut of thisspecies, some can enhance DENV infection in culture cell lines. But studies on miRNAthat play roles in DENV infection in the midgut of Ae. albopictus have not been carriedout, while previous studies indicate that although the midgut of Ae. albopictusis is moresusceptible to DENV infection than that of Ae. aegypti, subsequent dissemination ofDENV from the midgut is slower in Ae. albopictus than in Ae. aegypti, thus the miRNAinvolving in midgut infection by DENV should be different. Although previous studieshave identified some miRNA in Ae. albopictus, there is currently no availableinformation on the midgut miRNA of this species. Because the midgut is the first barrierto DENV infection, identifying the role of different midgut miRNA during the course ofinfection could aid the identification of vector-competent mosquitoes and therebyfacilitate the control of DENV-related diseases.In this study, mosquitoes Ae. albopictus, captured in wild places in Guangzhou, wereorally infected with DENV-2(NGC) and the midguts were dissected at differenttimepoints, midguts from sugar-fed and normal-blood-fed mosquitoes were alsoobtained as controls. Small RNA libraries from the migduts were constructed, highthroughput deep sequencing and bioinformatic analysis were used to obtain miRNAinformation in the midguts, novel miRNA were also predicted. Variation analysis,qRT-PCR and in vitro transient transfection assay were carried out subsequently to findmiRNA that may paly roles in in the process of DENV infection in the midgut of Ae.albopictus. Results from this study were listed as follows:1. The mosquitoes in this study were sensitive to DENV-2, the infection rate from themidguts was higher than that from the bodies (t=3.872, P=0.018). The infection rateincreased rapidly from3to7days post exposure (dpe) to DENV-2and reached morethan70%at14dpe.2. Types of miRNA in the midgut of Ae. albopictus were very abundant and somewere organ specific.112conserved miRNA represented by175sequences wereobtained and43novel miRNA were predicted. Besides, four miRNA, namely aal-miR-1174, aal-miR-2951, aal-miR-956-3p and aal-miR-956-5p, found in Ae.albopictus for the first time, were verified by stem loop qRT-PCR.3. miRNA expressing pattern changed greatly after blood meal. Comparing withsugar-fed group, expressing patterns of miRNA after blood meal changed greatly butcan be devided into senven types namely decreaded firstly and increase subsequently,decreased-increased-decreased, increased fastly and decreased fastly, increased steadlybut decreased slightly, increased continually, decreased continually and basically stable.Two types of patterns such as decreased-increased-decreased and decreaded-firstly andincrease-subsequently were the main changing patterns and separately account for32%and20%of all, several miRNAthat were highly expressed in the midgut belong to thesepatterns. After taking DENV-2blood meal, several miRNAchanged their normal pattern,for example, expressing levels of aal-miR-276-3p and aal-miR-275-3p decreased at24hpost normal blood meal but increased at the same timepoint post DENV-2infectedblood meal.4. Significant differences exist between infected and unfected midguts and types ofup-regulated miRNA were more than that of down-regulated ones.74miRNA wereexpressed with significant difference after infection,22were screened according to thefold-change and the expressing level,16were up-regulated including aal-miR-1767,-276-3p,-193-5p,-1951,-4728-5p,-6134,-622,-1420b-5p,-998-5p, and so on, and4were down-regulated such as aal-miR-1273f, aal-miR-4448, aal-miR-6666-3p andaal-miR-15-3p.5. Target genes of differently expressed miRNA were massive. In the absence ofgenome information for Ae. albopictus, the target genes of defferently expressedmiRNA were predicted on the genome of Ae. egypti,3608target genes were predictedand pathway analysis indicated that these genes were multifunctional. Also,43differently expressed miRNAcan find target sites on the genome of DENV-2.6. Four miRNAcan regulate replication of DENV-2in C6/36cells in our experiments.aal-miR-1767, aal-miR-4728-5p and aal-miR-276-3p enhanced replication of DENV-2in C6/36cells，aal-miR-4448inhibited replication of DENV-2in C6/36cells.Conclusions:1. This study presents the first study on miRNA specificly expressed in the midgut ofAe. albopictus, new types of conserved miRNA were found in mosquitoes for the firsttime and novel miRNA were predicted which contribute to miRNA studies inmosquitoes. 2. miRNA expressing pattern changed greatly after blood meal. Comparing with thatfrom sugar-fed mosquitoes, the expressing patterns changed greatly but can be devidedinto senven types namely decreaded firstly and increase subsequently,decreased-increased-decreased, increased fastly and decreased fastly, increased steadlybut decreased slightly, increased continually, decreased continually and basically stable.Two types of patterns including decreased-increased-decreased and decreased-firstlyand increased-subsequently were the main changing patterns and separately account for32%and20%of all, several miRNAthat were highly expressed in the midgut belong tothese patterns. After taking DENV-2blood meal, several miRNA changed their normalpattern which means they may play roles in midgut infection by DENV-2, for example,expressing levels of aal-miR-276-3p and aal-miR-275-3p decreased at24h post normalblood meal but increased at the same timepoint post DENV-2infected blood meal, thechanging means their potential rols in DENV-2infection.3. Difference analysis indicated that the changing tendencies of miRNA duringDENV-2infection were significant and were varied at different timepoints postinfection. most miRNA were up-regulated at24h but were down-regulated at48h postintaking DENV-2, the types of miRNA were the most and the amplitude of variationwere the biggest at7dpe.4. Results from an in vitro transient transfection assay indicate that some miRNA canregulate replication of DENV-2in C6/36cells, these miRNA should also play importantroles in midgut infection by DENV-2.