MOLECULAR CHARACTERIZATION OF DENGUE VIRUS HOST AND RESTRICTION FACTORS IN AEDES AEGYPTI MOSQUITOES

Despite decades of attempts at disease control, dengue remains one of the most significant mosquito-borne arboviral diseases, causing an estimated 390 million infections annually. While studies of molecular interactions between DENV and Ae. aegypti have paved a way for the development of alternative DENV control strategies, this field is still relatively understudied. Here, we used multiple molecular tools to study interactions between the virus and Ae. aegypti, as well as to identify DENV host and restriction factors. First, we have developed genetically modified mosquitoes with increased activity of the JAK/STAT pathway, and showed that these transgenic mosquitoes could inhibit DENV infection. Through microarray-based transcriptomic comparisons, we identified candidate DENV host and restriction factors and confirmed their function through RNAi. Second, we compared transcriptomic profiles of a panel of field-derived and laboratory Ae. aegypti strains with different DENV susceptibility. Through RNAi-mediated gene silencing, we have shown that basal level of immune activity, and expression level of host factors are important determinants for DENV susceptibility. Lastly, through a study of transcriptomic datasets comparing DENV-infected and uninfected Ae. aegypti, we identified and characterized lipid binding protein families, ML and NPC1, as host factors for DENV replication in Ae. aegypti

The Insect Microbiome Modulates Vector Competence for Arboviruses

Diseases caused by arthropod-borne viruses (arboviruses), such as Dengue, West Nile, and Chikungunya, constitute a major global health burden and are increasing in incidence and geographic range. The natural microbiota of insect vectors influences various aspects of host biology, such as nutrition, reproduction, metabolism, and immunity, and recent studies have highlighted the ability of insect-associated bacteria to reduce vector competence for arboviruses and other pathogens. This reduction can occur through mechanisms, such as immune response activation, resource competition, or the production of anti-viral molecules. Studying the interactions between insect vectors and their microbiota is an important step toward developing alternative strategies for arbovirus transmission control

Suppressing dengue-2 infection by chemical inhibition of Aedes aegypti host factors.

Dengue virus host factors (DENV HFs) that are essential for the completion of the infection cycle in the mosquito vector and vertebrate host represent potent targets for transmission blocking. Here we investigated whether known mammalian DENV HF inhibitors could influence virus infection in the arthropod vector A. aegypti. We evaluated the potency of bafilomycin (BAF; inhibitor of vacuolar H+-ATPase (vATPase)), mycophenolic acid (MPA; inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH)), castanospermine (CAS; inhibitor of glucosidase), and deoxynojirimycin (DNJ; inhibitor of glucosidase) in blocking DENV infection of the mosquito midgut, using various treatment methods that included direct injection, ingestion by sugar feeding or blood feeding, and silencing of target genes by RNA interference (RNAi). Injection of BAF (5 µM) and MPA (25 µM) prior to feeding on virus-infected blood inhibited DENV titers in the midgut at 7 days post-infection by 56% and 60%, and in the salivary gland at 14 days post-infection by 90% and 83%, respectively, while treatment of mosquitoes with CAS or DNJ did not affect susceptibility to the virus. Ingestion of BAF and MPA through a sugar meal or together with an infectious blood meal also resulted in various degrees of virus inhibition. RNAi-mediated silencing of several vATPase subunit genes and the IMPDH gene resulted in a reduced DENV infection, thereby indicating that BAF- and MPA-mediated virus inhibition in adult mosquitoes most likely occurred through the inhibition of these DENV HFs. The route and timing of BAF and MPA administration was essential, and treatment after exposure to the virus diminished the antiviral effect of these compounds. Here we provide proof-of-principle that chemical inhibition or RNAi-mediated depletion of the DENV HFs vATPase and IMPDH can be used to suppress DENV infection of adult A. aegypti mosquitoes, which may translate to a reduction in DENV transmission

Co-silencing of vATPase (vATP-V0B) and IMPDH.

<p>Co-silencing of vATPase (vATP-V0B) and IMPDH reduced DENV titers more than single silencing, although the difference between the co-silenced and vATPase single-silenced mosquito cohorts was not significant (p = 0.0535, Mann-Whitney test). ***; p<0.001, **; p<0.01, Mann-Whitney test. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p

Silencing of vATPase or IMPDH results in suppression of DENV infection.

<p>A–B) Silencing of vATPase subunit ac39 (vATP-ac39, AAEL011025), vATPase proteolipid subunit (vATP-V0B, AAEL012113), vATPase subunit f (vATP-f, AAEL002464), and vATPase 16-kDa proteolipid subunit (vATP-16, AAEL000291) reduced DENV titers in the Rock strain, C) in the SIN strain, and D) in the PTri strain. E) Silencing of inosine 5′-monophosphate dehydrogenase (IMPDH, AAEL009273) reduced DENV titers in the Rock strain. F) Silencing of vATPase (vATP-V0B) and IMPDH reduced DENV titers in salivary glands at 14 dpbm. ***; p<0.001, **; p<0.01, Mann-Whitney test. A–F) The red bar indicates a median value of titers. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p

Ingestion of BAF or MPA through the sugar or blood meal results in reduced DENV titers.

<p>BAF (50 µM) or MPA (250 µM) was mixed with 10% sucrose (A–B) or an infectious blood (C–D) and fed to mosquitoes. A) DENV titers at 7 dpbm of mosquitoes given BAF in a sugar meal were decreased when compared to the control group, but the difference was not significant (p = 0.0553). B) DENV titers at 7 dpbm of mosquitoes given MPA in a sugar meal were significantly reduced. C) Ingestion of BAF with an infectious blood meal reduced the DENV titers significantly. D) Ingestion of MPA with an infectious blood meal also reduced the titers significantly. (A–D) The red bar indicates a median value of titers. *; p<0.05, Mann-Whitney test. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p

Fitness impact of vATPase and IMPDH silencing.

<p>A) Mosquitoes were injected with vATPase dsRNA, IMPDH dsRNA and a longevity study was conducted. Survival was assessed until all mosquitoes had perished. The mean values from three replicates are shown, with the standard error bars. Additionally survival rates were analyzed by Kaplan-Meier survival analysis with Wilcoxon test, and detailed statistical information is available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003084#pntd.0003084.s005" target="_blank">Table S3</a>. B) Fecundity analysis was conducted after vATPase dsRNA, IMPDH dsRNA was injected. The horizontal bars represent the median value of eggs laid per female. ***; p<0.001, *; p<0.05, Mann-Whitney test. C) Fertility analysis was conducted with the eggs obtained from the fecundity assay. Hatch rates indicate the average percentage of eggs giving rise to first to second instar larvae. Mean values for hatch rates and standard errors (SE) of three biological replicates are indicated.</p

Injection of BAF or MPA after ingestion of virus does not suppress DENV infection.

<p>When BAF or MPA was injected 1 day after DENV infection, there was no reduction in DENV titers. P>0.05, Mann-Whitney test. A) Five micromolar BAF was injected into mosquitoes 1 day after DENV infection. B) Fifty micromolar MPA was injected 1 day after DENV infection. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p

Multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection.

Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models

Injection of BAF or MPA suppresses DENV infection in mosquito midguts.

<p>A) DENV titers of bafilomycin (BAF; 5 µM and 25 µM)-injected mosquito midguts at 7 days post-blood meal (dpbm) were compared to those of the DMSO-injected control group. Each data point represents virus infection intensity (titer) from an individual midgut. Each mosquito was injected with 0.345 pmole (214.9 pg) or 1.725 pmole (1.1 ng) of BAF. B) DENV titers of mycophenolic acid (MPA; 50 µM and 250 µM)-injected mosquito midguts at 7 dpbm were compared to those of the DMSO-injected control group. Each mosquito was injected with 3.45 pmole (1.1 ng) or 17.25 pmole (5.5 ng) of MPA. C) DENV titers of mosquitoes injected with either 5 µM BAF, 250 µM MPA, or a cocktail of both compounds were compared to those of the control group. D–E) DENV titers of castanospermine (CAS, 250 mM and 1 mM)- or deoxynojirimycin (DNJ, 250 mM and 1 mM)-injected mosquito midguts at 7 dpbm were compared to those of the control group. F) DENV titers of BAF (5 µM) and MPA (250 µM)-injected mosquito salivary glands at 14 dpbm were reduced compared to those of the DMSO-injected control group. A–F) The red bar indicates a median value of titers. A–B, D–F) Mann-Whitney test. C) Kruskal-Wallis test. ***; p<0.001, **; p<0.01, *; p<0.05. Descriptive statistics for DENV infection assays are presented in supplementary table S2.</p