Mosquito Saliva Modulates Japanese Encephalitis Virus Infection in Domestic Pigs

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is the leading cause of pediatric viral encephalitis in Asia. Japanese encephalitis virus is transmitted by Culex species mosquitoes that also vector several zoonotic flaviviruses. Despite the knowledge that mosquito saliva contains molecules that may alter flavivirus pathogenesis, whether or not the deposition of viruses by infected mosquitoes has an impact on the kinetics and severity of JEV infection has not been thoroughly examined, especially in mammalian species involved in the enzootic transmission. Most JEV pathogenesis models were established using needle inoculation. Mouse models for West Nile (WNV) and dengue (DENV) viruses have shown that mosquito saliva can potentiate flavivirus infections and exacerbate disease symptoms. In this study, we determined the impact of mosquito salivary components on the pathogenesis of JEV in pigs, a species directly involved in its transmission cycle as an amplifying host. Interestingly, co-injection of JEV and salivary gland extract (SGE) collected from Culex quinquefasciatus produced milder febrile illness and shortened duration of nasal shedding but had no demonstrable impact on viremia and neuroinvasion. Our findings highlight that mosquito salivary components can differentially modulate the outcomes of flavivirus infections in amplifying hosts and in mouse models.

Assessment of fitness and vector competence of a New Caledonia wMel Aedes aegypti strain before field-release

Background Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. Methods/principal findings The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. Conclusions/significance Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC.

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.

Type I hypersensitivity promotes Aedes aegypti blood feeding

Mosquitoes play a major role in human disease by serving as vectors of pathogenic microorganisms. Mosquitoes inject saliva into host skin during the probing process. Mosquito saliva contains a number of proteins that facilitate blood feeding by preventing hemostasis. Mosquito saliva also contains potent allergens that induce type I hypersensitivity reactions in some individuals. Type I hypersensitivity reactions in skin involve IgE-mediated degranulation of mast cells, which leads to vasodilation and an itch sensation. We hypothesized that hypersensitivity to mosquito saliva influences blood feeding. To test this hypothesis, we recruited human subjects who consented to Aedes aegypti bites. We measured their first sensation of itch, the strength of their itch sensation, the number of times mosquitoes attempted to feed, the number of times mosquitoes probed their skin, feeding time, engorgement status, and wheal diameter. Here we show that hypersensitive subjects had a stronger itch sensation, and that the time to first itch sensation was inversely correlated with wheal diameter; however, mosquitoes tended to probe less and engorge more on these subjects. Follow-up experiments testing the impact of oral antihistamine treatment on mosquito feeding parameters failed to reveal a statistically significant result. Histamine also failed to promote blood feeding on an artificial membrane feeder. This study suggests that mosquito saliva-induced type I hypersensitivity promotes blood feeding but that this may be independent from histamine or histamine signaling.

A laboratory-based study to explore the use of honey-impregnated cards to detect chikungunya virus in mosquito saliva

Mosquito control is implemented when arboviruses are detected in patients or in field-collected mosquitoes. However, mass screening of mosquitoes is usually laborious and expensive, requiring specialized expertise and equipment. Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito. Here we test the use of filter papers to detect chikungunya virus in mosquito saliva in laboratory conditions, before proposing this method in large-scale mosquito surveillance programs. We found that 0.9 cm2 cards impregnated with a 50% honey solution could replace the forced salivation technique as they offered a viral RNA detection until 7 days after oral infection of Aedes aegypti and Aedes albopictus mosquitoes with CHIKV.

The anti-immune dengue subgenomic flaviviral RNA is found in vesicles in mosquito saliva and associated with increased infectivity

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We show that salivary sfRNA is protected in detergent-sensitive, protease-resistant compartments. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in human cells. Since sfRNA potently inhibits innate immunity in human cells, we posit that sfRNA in mosquito saliva is present in extracellular vesicles that deliver it to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

Mosquito saliva sialokinin-dependent enhancement of arbovirus infection through endothelial barrier leakage

SummaryViruses transmitted by Aedes mosquitoes constitute an increasingly important global health burden. Defining common determinants of host susceptibility to this large group of heterogenous pathogens are key for informing the rational design of new pan-viral medicines. Infection of the vertebrate host with these viruses is enhanced by the presence of mosquito saliva, a complex mixture of salivary gland-derived factors and microbiota. We show that enhancement of infection by saliva was dependent on vascular function and was independent of most anti-saliva immune responses, including to salivary microbiota. Instead, the Aedes gene product sialokinin mediated enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having vertebrate-like tachykinin sequence and is absent from non-vector competent Anopheles mosquitoes, whose saliva was not pro-viral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have potential to limit disease severity following infection with Aedes mosquito-borne viruses.

A mosquito salivary protein facilitates sporozoite traversal of host cells

Malaria begins when Plasmodium-infected Anopheles mosquitoes take a blood meal on a vertebrate. During the initial probing process, mosquitoes inject saliva and sporozoites into the host skin. Components of mosquito saliva have the potential to influence sporozoite functionality. Sporozoite-associated mosquito saliva protein 1 (SAMSP1, AGAP013726) was among several proteins identified when sporozoites were isolated from saliva, suggesting it may have an effect on Plasmodium. Recombinant SAMSP1 enhanced sporozoite gliding and cell traversal activity in vitro. Moreover, SAMSP1 decreased neutrophil chemotaxis in vivo and in vitro, thereby also exerting an influence on the host environment in which the sporozoites reside. Active or passive immunization of mice with SAMSP1 or SAMSP1 antisera diminished the initial Plasmodium burden following infection. Passive immunization of mice with SAMSP1 antisera also added to the protective effect of a circumsporozoite protein (CSP) monoclonal antibody. SAMSP1 is, therefore, a mosquito saliva protein that can influence sporozoite infectivity in the vertebrate host.