Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.     

Evolution of endemic and sylvatic lineages of dengue virus

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.     

A network-patch methodology for adapting agent-based models for directly transmitted disease to mosquito-borne disease

Mosquito-borne diseases cause significant public health burden and are widely re-emerging or emerging. Understanding, predicting, and mitigating the spread of mosquito-borne disease in diverse populations and geographies are ongoing modelling challenges. We propose a hybrid network-patch model for the spread of mosquito-borne pathogens that accounts for individual movement through mosquito habitats, extending the capabilities of existing agent-based models (ABMs) to include vector-borne diseases. The ABM are coupled with differential equations representing ‘clouds’ of mosquitoes in patches accounting for mosquito ecology. We adapted an ABM for humans using this method and investigated the importance of heterogeneity in pathogen spread, motivating the utility of models of individual behaviour. We observed that the final epidemic size is greater in patch models with a high risk patch frequently visited than in a homogeneous model. Our hybrid model quantifies the importance of the heterogeneity in the spread of mosquito-borne pathogens, guiding mitigation strategies.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.     

Strategies for introducing Wolbachia to reduce transmission of mosquito-borne diseases

Certain strains of the endosymbiont Wolbachia have the potential to lower the vectorial capacity of mosquito populations and assist in controlling a number of mosquito-borne diseases. An important consideration when introducing Wolbachia-carrying mosquitoes into natural populations is the minimisation of any transient increase in disease risk or biting nuisance. This may be achieved by predominantly releasing male mosquitoes. To explore this, we use a sex-structured model of Wolbachia-mosquito interactions. We first show that Wolbachia spread can be initiated with very few infected females provided the infection frequency in males exceeds a threshold. We then consider realistic introduction scenarios involving the release of batches of infected mosquitoes, incorporating seasonal fluctuations in population size. For a range of assumptions about mosquito population dynamics we find that male-biased releases allow the infection to spread after the introduction of low numbers of females, many fewer than with equal sex-ratio releases. We extend the model to estimate the transmission rate of a mosquito-borne pathogen over the course of Wolbachia establishment. For a range of release strategies we demonstrate that male-biased release of Wolbachia-infected mosquitoes can cause substantial transmission reductions without transiently increasing disease risk. The results show the importance of including mosquito population dynamics in studying Wolbachia spread and that male-biased releases can be an effective and safe way of rapidly establishing the symbiont in mosquito populations.     

Infecting mosquitoes alters DENV-2 characteristics and enhances hemorrhage-induction potential in Stat1-/- mice

Dengue is one of the most prevalent arthropod-borne viral diseases in humans. There is still no effective vaccine or treatment to date. Previous studies showed that mosquito-derived factors present in saliva or salivary gland extract (SGE) contribute to the pathogenesis of dengue. In this study, we aimed to investigate the interplay between mosquito vector and DENV and to address the question of whether the mosquito vector alters the virus that leads to consequential disease manifestations in the mammalian host. DENV2 cultured in C6/36 cell line (culture-DENV2) was injected to Aedes aegypti intrathoracically. Saliva was collected from infected mosquitoes 7 days later. Exploiting the sensitivity of Stat1^-/- mice to low dose of DENV2 delivered intradermally, we showed that DENV2 collected in infected mosquito saliva (msq-DENV2) induced more severe hemorrhage in mice than their culture counterpart. Msq-DENV2 was characterized by smaller particle size, larger plaque size and more rapid growth in mosquito as well as mammalian cell lines compared to culture-DENV2. In addition, msq-DENV2 was more efficient than culture-DENV2 in inducing Tnf mRNA production by mouse macrophage. Together, our results point to the possibility that the mosquito vector provides an environment that alters DENV2 by changing its growth characteristics as well as its potential to cause disease.     

Glyphosate inhibits melanization and increases susceptibility to infection in insects

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin’s broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate—the most widely used herbicide globally—inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate’s mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation–reduction balance. Overall, these findings suggest that glyphosate’s environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Glyphosate, the most commonly used herbicide in the world, inhibits the production of melanin. Melanin is an important pigment and a key component of the insect immune system; this study shows that glyphosate weakens insects’ melanin-based immune system and makes them more vulnerable to infections, including with the human malaria parasite Plasmodium falciparum.     

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.     

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.     

Innate immune response to Rift Valley fever virus in goats

Rift Valley fever (RVF), a re-emerging mosquito-borne disease of ruminants and man, was endemic in Africa but spread to Saudi Arabia and Yemen, meaning it could spread even further. Little is known about innate and cell-mediated immunity to RVF virus (RVFV) in ruminants, which is knowledge required for adequate vaccine trials. We therefore studied these aspects in experimentally infected goats. We also compared RVFV grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Goats developed viremia one day post infection (DPI), which lasted three to four days and some goats had transient fever coinciding with peak viremia. Up to 4% of peripheral blood mononuclear cells (PBMCs) were positive for RVFV. Monocytes and dendritic cells in PBMCs declined possibly from being directly infected with virus as suggested by in vitro exposure. Infected goats produced serum IFN-γ, IL-12 and other proinflammatory cytokines but not IFN-α. Despite the lack of IFN-α, innate immunity via the IL-12 to IFN-γ circuit possibly contributed to early protection against RVFV since neutralising antibodies were detected after viremia had cleared. The course of infection with insect cell-derived RVFV (IN-RVFV) appeared to be different from mammalian cell-derived RVFV (MAM-RVFV), with the former attaining peak viremia faster, inducing fever and profoundly affecting specific immune cell subpopulations. This indicated possible differences in infections of ruminants acquired from mosquito bites relative to those due to contact with infectious material from other animals. These differences need to be considered when testing RVF vaccines in laboratory settings.     

Dengue virus-like particles: construction and application

Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.     

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus‐2 and inhibits viral replication

Dengue virus (DENV) comprises of four serotypes (DENV‐1 to ‐4) and is medically one of the most important arboviruses (arthropod‐borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV‐2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti‐lachesin antibody resulted in a significant reduction in DENV replication.     

Managing mosquitoes without destroying wetlands: an eastern Australian approach

Recognising both the importance of intertidal wetlands and their role in mosquito-borne disease we discuss wise management to conserve wetland values and to reduce vector borne disease health risks. First we summarise the mosquito-borne diseases associated with intertidal wetlands in sub-tropical and tropical Australia. We consider the Ramsar Strategic Plan, its reflection in some key Australian statutes and the relationship between environment-focussed legislation and health legislation. This is followed by a brief overview of mosquito control and its impact on human health. Using a salt marsh example of an integrated process, we describe the development of what was, in the 1980s in Australia, a novel method of habitat modification (runnelling) for mosquito control. Runnelling modifies the tidal water flow on salt marshes, reducing mosquito larval numbers and minimising environmental impacts. The approach is related to two of the Ramsar goals (wise use and institutional capacity and effectiveness). We then describe the extension of its rationale to a complex mangrove system. Finally, with a concept model, we consider the convergence between minimal habitat modification for wetland conservation and human health protection using an interdisicplinary approach involving multiple stakeholders.     

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.     

A plant pathogen modulates the effects of secondary metabolites on the performance and immune function of an insect herbivore

Host plant chemical composition critically shapes the performance of insect herbivores feeding on them. Some insects have become specialized on plant secondary metabolites, and even use them to their own advantage such as defense against predators. However, infection by plant pathogens can seriously alter the interaction between herbivores and their host plants. We tested whether the effects of the plant secondary metabolites, iridoid glycosides (IGs), on the performance and immune response of an insect herbivore are modulated by a plant pathogen. We used the IG‐specialized Glanville fritillary butterfly Melitaea cinxia, its host plant Plantago lanceolata, and the naturally occurring plant pathogen, powdery mildew Podosphaera plantaginis, as model system. Pre‐diapause larvae were fed on P. lanceolata host plants selected to contain either high or low IGs, in the presence or absence of powdery mildew. Larval performance was measured by growth rate, survival until diapause, and by investment in immunity. We assessed immunity after a bacterial challenge in terms of phenoloxidase (PO) activity and the expression of seven pre‐selected insect immune genes (qPCR). We found that the beneficial effects of constitutive leaf IGs, that improved larval growth, were significantly reduced by mildew infection. Moreover, mildew presence downregulated one component of larval immune response (PO activity), suggesting a physiological cost of investment in immunity under suboptimal conditions. Yet, feeding on mildew‐infected leaves caused an upregulation of two immune genes, lysozyme and prophenoloxidase. Our findings indicate that a plant pathogen can significantly modulate the effects of secondary metabolites on the growth of an insect herbivore. Furthermore, we show that a plant pathogen can induce contrasting effects on insect immune function. We suspect that the activation of the immune system toward a plant pathogen infection may be maladaptive, but the actual infectivity on the larvae should be tested.     

Reproductive phase locking of mosquito populations in response to rainfall frequency

The frequency of moderate to heavy rainfall events is projected to change in response to global warming. Here we show that these hydrologic changes may have a profound effect on mosquito population dynamics and rates of mosquito-borne disease transmission. We develop a simple model, which treats the mosquito reproductive cycle as a phase oscillator that responds to rainfall frequency forcing. This model reproduces observed mosquito population dynamics and indicates that mosquito-borne disease transmission can be sensitive to rainfall frequency. These findings indicate that changes to the hydrologic cycle, in particular the frequency of moderate to heavy rainfall events, could have a profound effect on the transmission rates of some mosquito-borne diseases.