Dengue virus infection mosquito

They then analyzed the resultant high-resolution videos using computer software that broke down the various elements of mosquito behavior and biting pattern into precise activities. Subsequent experimental and statistical analyses revealed that both of these changes tripled transmission efficiency. More specifically, the researchers found that infected mosquitos are significantly less able to locate a blood vessel to feed from with the first insertion of their probe.

Instead, they have to insert and re-insert the probe until they are successful. Dengue is a mosquito-borne disease that affects more than million people each year worldwide, killing around 40, For most people, infection causes no symptoms or mild disease such as nausea, vomiting, rash, fever, and aches and pains.

However, one in 20 infected people develops severe dengue, which can lead to shock, internal bleeding, and death. The research team next want to understand the molecular mechanisms behind these changes to mosquito behavior. If scientists can identify a gene or protein responsible for the changes, they might be able to design chemicals to alter them.

Source: Duke-NUS. Watkins Graduate Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. With 2. It exists as four closely related but antigenically distinct serotypes DENV-1, -2, -3, and -4 , all of which have Aedes aegypti mosquitoes as their primary vector, with A.

The incidence and geographic range of dengue and dengue hemorrhagic fever have increased dramatically in recent decades, and since there is at present no licensed vaccine or drug treatment against DENV, vector control remains the best method for preventing transmission. Although vertical transmission of the virus has been reported [2] , [3] , mosquitoes mainly acquire DENV by feeding on the blood of an infected human.

DENV first infects and replicates in the mosquito midgut epithelium. It subsequently spreads through the hemolymph to replicate in other organs such as the fat body and trachea, finally infecting the salivary gland at approximately 10—14 days post-bloodmeal [4].

Once in the saliva, DENV can be inoculated into a human host when the mosquito acquires a blood meal, thus spreading the disease. The mosquito salivary gland plays important roles in DENV transmission.

Firstly, infection of the gland itself is an essential part of the transmission cycle. Secondly, the salivary gland produces numerous anti-coagulant, anti-inflammatory and vasodilatory molecules which facilitate probing and bloodmeal acquisition [5] — [10] , as well as immune factors that reduce microbial loads in ingested blood and nectar.

Lastly, mosquito saliva can impair the immune response of the vertebrate host to arbovirus infection, resulting in increased viremia levels and increasing the risk of virus transmission reviewed in [11]. Despite its importance in pathogen transmission, the current knowledge on antiviral defense in the salivary gland is limited and is mainly represented by a recent study which identified a cecropin-like peptide with antibacterial and antiviral activities that was induced upon DENV infection of the gland [12].

Mosquitoes are exposed to a variety of microbes in their natural habitats, and possess an innate immune system capable of mounting a potent response against microbial challenge. To date, however, most studies of mosquito antiviral immunity have examined DENV replication in the midgut, but not in other biologically relevant compartments such as the salivary gland. In addition, despite the well-documented involvement of the Toll and JAK-STAT pathways in insect immunity, the specific molecular mechanisms by which these pathways act remain uncharacterized.

Viral pathogen-associated molecular patterns PAMPs and their associated insect pattern recognition receptors PRRs have not yet been discovered, and only a few putative antiviral effector molecules have been identified [12] , [15] — [17]. To gain a better understanding of how the A. These experiments revealed intriguing patterns of differential transcript abundance that suggested a broad impact of DENV infection on a variety of salivary gland functions, including those implicated in immunity, host-seeking, and blood acquisition.

To confirm the functional relevance of DENV-modulated transcript abundance, we used an RNAi-mediated gene silencing approach to show that three DENV infection-induced salivary gland-enriched transcripts can modulate DENV replication in the salivary gland, corroborating the earlier finding [12] that this organ mounts an anti-viral response. In addition, we show for the first time that silencing of two DENV infection-induced odorant-binding protein OBP transcripts impaired the host-seeking and blood-feeding ability of mosquitoes, suggesting that the virus is capable of modifying mosquito behavior through the regulation of chemosensory genes.

Finally, inspired by these findings, we extended our study to show that DENV is likely to exert a broader impact on mosquito chemosensation by infecting its main olfactory organs, the antennae. To determine the A. We reasoned that this analysis would yield information about potential gene function, since salivary gland-enriched transcripts would be more likely to perform functions specific to this organ.

Of the total number of salivary gland-expressed transcripts, The transcripts of genes Differentially expressed transcripts are presented in Table S1. A previous study by Ribeiro et al. Our study detected the vast majority out of of these transcripts, supporting the robustness and validity of our microarray-based approach.

B Salivary gland-expressed genes were classified into low, medium and high abundance categories according to their spot intensities on the array. The pie chart shows the number of genes in each category and its corresponding percentage of the total number of expressed genes.

D Venn diagram showing numbers of uniquely and commonly regulated genes in DENV-infected salivary glands and carcasses. Arrows represent the direction of gene regulation. Since our microarray-based analyses only provide information on the ratio of differential transcript abundance between the compared samples, we also considered the absolute abundance levels of salivary gland transcripts. Based on the fluorescence intensity of their spots on the microarray, we categorized transcripts into high, medium, and low abundance Table S2.

This distribution is comparable to what has been observed for the Anopheles gambiae salivary gland transcriptome under the same analysis [6]. Genes that displayed differential transcript abundance between the salivary gland and the carcass represented a range of functional classes Figure 1C.

We next provide a brief description of several functional classes that we consider pertinent to salivary gland function. Several protein digestive enzyme transcripts, including 12 trypsins, an amidase, a serine protease and an endopeptidase, were enriched in the salivary gland.

These enzymes in saliva could be ingested along with the bloodmeal and aid its digestion. Alternatively, they could be involved in proteolytic events that occur in the vertebrate host during blood-feeding, such as clot prevention or digestion of extra-cellular matrix components [6]. These included two MD2-like proteins, six fibrinogen-related proteins, eight antimicrobial peptides AMPs , 27 serine proteases, and several Toll pathway-related genes three Spaetzles and two Tolls.

Although nothing is known about the potential roles of these genes in salivary gland immunity, their enrichment in this organ suggests that the gland is capable of mounting potent and diverse immune responses against pathogen challenge to ensure sterility of ingested blood and nectar. AMPs may act against bacteria that the mosquito comes into direct contact with during feeding, for example in sugar sources or on vertebrate skin.

The large number of salivary gland-enriched serine protease transcripts is striking; these could be implicated in immune pathway activation through the triggering of serine protease cascades, or some of these may play roles in blood-feeding by hydrolyzing host proteins to prevent clotting or inflammation [7]. Blood-feeding activates vertebrate host responses that inhibit blood flow, activate antimicrobial defenses, or call the attention of the host to the feeding mosquito.

For example, ATP and ADP released from injured cells and activated platelets at the feeding site stimulate platelet aggregation and mast cell degranulation, and adenosine activates inflammatory responses that result in itching and burning sensations, increasing the likelihood of detection of the mosquito [7] , [8].

To counteract these responses, mosquito saliva contains numerous enzymes that perform anti-hemostatic and anti-inflammatory roles. Thrombin is a key enzyme in the vertebrate blood coagulation cascade.

Two putative anti-thrombin transcripts AAEL, AAEL were salivary gland-enriched and also belonged to the highly abundant transcript category.

AAEL encodes a Kazal-type serine protease inhibitor that has been partially characterized and found to have anticoagulant and thrombin inhibitory activity [22]. Transcripts of the D7 gene family are widely found in mosquito sialotranscriptomes [7].

D7 protein family members have been suggested to bind and sequester biogenic amines such as serotonin, histamine, norepinephrine and epinephrine [23] , which are released at the site of injury and play roles in platelet aggregation, vasoconstriction, and inflammation. Four D7 family member transcripts one — AAEL — belonged to the highly abundant category were found to be enriched in the salivary gland.

The D7 proteins are related to the odorant-binding proteins OBPs , and may have been co-opted from this family to scavenge biogenic amines [7] , [23]. The vast majority of these transcripts belonged to the low abundance category, with the exceptions of AAEL encoding a conserved hypothetical protein with a predicted role in odorant binding and AAEL encoding OBP11 , which belonged to the high abundance category.

OBPs are small, water soluble, secreted proteins that are highly abundant in the lymph of the sensilla of insect antennae and maxillary palps. They are specialized for ligand binding, and are thought to act as carriers for hydrophobic odorant molecules by facilitating their transport through the aqueous lymph to OR neurons [24] , [25]. Enrichment of their transcripts in the salivary gland was somewhat unexpected, but salivary gland OBP transcripts have been reported in A.

DENV altered the abundance of 38 transcripts with functions related to metabolic processes, transport and stress response. Six transcripts with predicted cytoskeletal functions were enriched upon infection; this may reflect maintenance in structural integrity of the infected salivary gland, since cytopathology has been reported in this organ following arbovirus infection [28] , [29].

Also up-regulated were two tetraspanin transcripts, which encode transmembrane proteins that have roles in cell-cell interactions, adhesion, motility, and proliferation. Twelve transcripts with immune-related functions were induced by DENV infection, and included two MD2-like gene family members, which code for secreted proteins containing Niemann-Pick lipid recognition domains.

These data suggest a potential role for A. Transcripts encoding a transferrin and a fibrinogen-related protein were also up-regulated. Transferrins bind iron with high affinity and play roles in iron metabolism, immunity, and development.

They are up-regulated upon parasite or bacterial infection, and may sequester iron from pathogens; alternatively, proteolytic fragments from these proteins have also been suggested to also act as anti-microbial peptides or inducers of the immune response [34].

Fibrinogen-related proteins bind bacteria and parasites in mosquitoes and may function as pattern recognition receptors [35]. Transcripts of three leucine-rich repeat LRR -containing proteins and one ankyrin repeat-containing protein were induced by DENV infection.

The broader LRR-containing protein family includes the mosquito Tolls, and family members are commonly involved in protein-protein interactions and signal transduction pathways [36].

Ankyrin repeats mediate protein-protein interactions and are present in several immune-related proteins, such as the IkB inhibitory domain of the NFkB-like transcription factor Rel2 of the mosquito IMD immune pathway. Cathepsin Bs are lysosomal cysteine proteases known to be involved in the apoptosis of immune cells [37]. They can also play roles in TLR signaling, and are required to cleave the endolysosomal TLRs 7 and 9 before these molecules can signal [38] — [40].

Cystatins are cysteine protease inhibitors that may play roles in regulating apoptosis, since many enzymes such as the caspases and cathepsins involved in apoptotic pathways are cysteine proteases [41] , [42]. A cystatin has also been reported to induce autophagy in mammalian cells [43] , and DENV is known to induce autophagy as a means of regulating lipid metabolism in the host cell [44] , [45]. The transcripts of three peptides with sequence similarity to secreted salivary peptides from Culicine mosquito species were also up-regulated by DENV infection.

The functions of these peptides remain unknown, but some may be involved in the production of allergic reactions to mosquito bites [7].

To determine whether our observed salivary gland transcriptomic infection responses were specific for this organ, or if they also occurred in other tissues, we went on to characterize the DENV infection-responsive carcass transcriptome at 14 dpbm.

Only 28 genes were similarly regulated between the salivary gland and the carcass upon infection Figure 1D , indicating that the transcriptomic responses in these two compartments are quite distinct. Our transcriptomic analyses suggested that at least some of the DENV infection-responsive transcripts may play roles in limiting infection, or reflect a virus-mediated modulation of salivary gland functions that could have implications for mosquito behavior.

We were particularly interested in modulators of DENV replication in the salivary gland. Based on our transcriptomic analyses and literature searches, we selected seven candidate genes for functional analysis via RNAi-mediated gene silencing Table 1.

We have elaborated on the potential modes of action of these genes in the previous section. Mosquitoes were orally infected with DENV through a bloodmeal, and candidate genes were silenced in the salivary gland at 7 dpbm by the injection of 2 ug of dsRNA per mosquito [19]. At this time point, the midgut and carcass are fully infected, and the virus is initiating infection of the salivary gland [4].

Salivary glands were subsequently dissected at 7 days post-silencing 14 dpbm , and virus titers were determined by plaque assay. Candidate genes were silenced in DENV-infected mosquitoes, and salivary gland virus titers at 14 dpbm were determined by plaque assay. Since injection of dsRNA into the mosquito thorax results in non-compartment-specific silencing, it is possible that the altered virus titers observed in the salivary gland are a consequence of gene silencing in other parts of the mosquito carcass.

However, we consider this less likely for several reasons: firstly, DENV infection induced these genes only in the salivary gland and not in the carcass Table 1 , suggesting that they play infection-related functions in the gland; secondly, dsRNA injections were carried out at 7 dpbm, when carcass DENV titers have already peaked, while salivary gland infection is just beginning [4] ; and lastly, we found no significant differences in virus titers between the carcasses of DENV-infected gene-silenced and control GFP dsRNA-treated mosquitoes Figure S2.

This finding was unexpected and intriguing to us, and we hypothesized that these genes could participate in chemosensory signaling during host-seeking or probing. To test this hypothesis, these genes were individually silenced by the injection of 2 ug dsRNA per mosquito, 4 days prior to a behavioral feeding assay.

Mosquitoes were offered an anesthetized Swiss Webster mouse, and the following parameters were measured: a Probing propensity percentage of mosquitoes that probed within a fixed time period ; b Probing initiation time time from the introduction of the mouse to the time at which the mosquito initiated probing — a rough measure of host-seeking ability ; and c Probing time time from the initial insertion of the proboscis in the skin to the initial engorgement of blood [6] , [10].

Probing time was also increased in OBP gene-silenced mosquitoes, although this increase was not statistically significant Figure 3C.

Since only mosquitoes that probed were considered for the probing time analysis, the lower number of mosquitoes that probed in OBP-silenced groups could have contributed to the lack of statistical significance for this parameter. Taken together, these data indicate that gene silencing of these OBPs impairs the efficiency of mosquito blood-feeding. A behavioral feeding assay was performed 4 days post-silencing of OBPs 10 and Mosquitoes were offered an anesthetized mouse and observed individually for seconds.

The following parameters were measured and are represented here: A Probing propensity: the percentage of mosquitoes that probed within the observation period; B Probing initiation time: the time from the introduction of the mouse until the mosquito starts to probe; C Probing time: the time from the initial insertion of the mouthparts in the skin to the initial engorgement of blood. The observed effect on feeding behavior could also be due to gene silencing in the chemosensory organs antennae and maxillary palps instead of or in addition to the salivary gland.

To further investigate the molecular basis of this interesting phenotype, we determined OBP gene silencing efficiency in these two body compartments by quantitative RT-PCR. High silencing efficiencies for both OBP genes were consistently obtained in the salivary gland averages of While these data suggest that the impaired feeding behavior was at least in part due to OBP gene silencing in the salivary gland, we also considered the possibility that the altered host-seeking and feeding behavior was due to DENV infection of the antennae and its effect on OBP transcript abundance there.

To test the hypothesis that DENV infects the antennae and as such can influence OBP transcript abundance, immunofluorescent staining was first performed on head squashes of orally-infected mosquitoes at 14 dpbm. DENV-infected cells were clearly present in the antennae of infected mosquitoes but not in mock-infected controls Figure 5. Female A. Relative DENV loads were significantly higher at 14 dpbm compared to 10 dpbm, indicating that virus actively replicates in the chemosensory organs Figure 4B.

Head squashes from mosquitoes at 14 dpbm were stained with mouse hyperimmune ascitic fluid to DENV and an AlexaFluorconjugated anti-mouse antibody. Red, DENV antigen. We next compared OBP transcript abundance in the chemosensory organs antennae, palps and proboscis of DENV- and mock-infected mosquitoes.

Insect ORs form heteromeric complexes consisting of a conventional OR and a highly conserved universal co-receptor, termed OR co-receptor Orco [46]. We found that A. The behavioral and gene expression data presented above suggest that DENV infection may heighten the chemosensory abilities of mosquitoes, rendering them more efficient at bloodmeal acquisition.

To test this hypothesis, we compared the blood-feeding behavior of DENV- and mock-infected mosquitoes at 14 dpbm. Workers at risk include: Outdoor workers Business travelers who may travel to areas with mosquito-borne diseases Laboratory workers who may work with potentially infected samples, cultures, or arthropods Healthcare workers who may handle patients who are, or might be infected with certain mosquito-borne diseases.

Transmission may occur through a break in their skin or via a sharp penetration injury. Employer Recommendations Some mosquitoes lay eggs in or near standing water.

Decrease the numbers of mosquitoes at worksites by: removing, turning over, covering, or storing equipment removing debris from ditches filling in ruts and other areas that collect standing water removing tires, buckets, bottles, and barrels that collect water placing drain holes in containers that collect water and cannot be discarded Keep mosquitoes outside by ensuring that doors and windows have screens and are kept closed when possible.

Provide training about: the risk of mosquito bites and how to prevent them symptoms of mosquito-borne diseases the safe use of insect repellents Provide workers with, and encourage them to wear, clothing that covers theirs hands, arms, legs, and other exposed skin. Consider providing hats with mosquito netting to protect the face and neck.

When used as directed, EPA-registered insect repellents are proven safe and effective, even for pregnant and breastfeeding women.

If also using sunscreen, apply sunscreen first and insect repellent second. Permethrin can be applied to clothing and gear. Wear clothing that covers hands, arms, legs, and other exposed skin. Wear hats with mosquito netting to protect the face and neck.

Remove standing water for example, tires, buckets, barrels to reduce places where mosquitoes lay eggs Workers who develop symptoms of a mosquito-borne disease should report this promptly to their supervisor and get medical attention.

West Nile Virus.