Wolbachia-mediated Mosquito vector control against deadly arboviruses

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By [Kye Duren]

Aedes Mosquitoes: Vectors for the Ages

What Is a disease vector

According to the World Health organization (WHO), a disease vector is a living organism that has the ability to spread infectious diseases between human, animals, and animals and humans [21]. The most successful disease vectors are bloodsucking organisms, like ticks, sandflies, fleas, and mosquitoes, which can both receive and transmit diseases through blood meals [21]. WHO also states that vector-borne diseases account for 17% of all infectious diseases. At the same time theses vector-borne diseases also account for 1 million deaths annually [21].

Aedes Mosquitoes

Mosquitoes are currently the best known system for vectoring infectious diseases. Arguably the most notorious of these disease vectors hail from the genus Aedes [21]. Aedes mosquitoes are distinguishable by black and white markings on their abdomen, thorax, and legs. Aedes female mosquitoes take blood meals by biting humans and domesticated animals, ingesting their blood, and using it as nutrients to lay eggs. It is in this exchange that dangerous vector-borne diseases can be transmitted from both host to mosquito and from mosquito to host. The mechanism by which Aedes mosquitoes become infected with arboviruses is through the uptake of infected blood. Non-mosquito host infection usually occurs during a mosquito blood meal when the mosquito vector secretes infected saliva into the host body. Aedes mosquito saliva contains molecules that limit blood clotting and inflammation, but enhance modulation of the human immune response. At the same time mosquito saliva may cause a decrease in antiviral response as well allowing viral infection to take effect. In addition, unlike other mosquito genus’s, these mosquitoes are active daytime biters, which give them a higher chance of interacting with humans. Aedes aegypti and Aedes albopictus mosquitoes are the premier vectors within the genus, making them some of the best disease vectors in the world.


Aedes Aegypti

Aedes aegypti feeding in Dar es Salaam, Tanzania. By Muhammad Mahdi Karim [1]

Aedes aegypti (Ae.ae) mosquitoes are dark-colored mosquitoes with characteristic white lyre- shaped markings and legs incorporating both patterns [17]. Female Ae.ae are daytime mosquitoes, being most active immediately after sunrise and a few hours before sunset [17]. These mosquitoes are stealthy biters as they tend to take blood meals from ankles and elbows, and approach from behind the host [15]. Three days after taking a blood meal, Ae.ae females can lay their eggs. Ae.ae females lay eggs in water natural or artificial water containing vessels above the level of the water [15]. Larvae hatch when rainwater covers the egg [15]. Larvae then undergo the aquatic life cycle to adulthood over the next 7-9 days [15]. Mosquito eggs are quite durable, as they can survive for about 6 months in a dormant state without water. However Ae.ae mosquitoes and their eggs can’t survive in cold temperatures or through the winter [15].Ae.ae are endemic to Africa, but through international shipping routes, the mosquito is now found in most tropical and subtropical regions. Ae.ae mosquitoes are disease vectors of Dengue virus (DENV), Chikungunya virus (CHIKV), and Yellow Fever, but they have also vectored West Nile virus and Malaria [19].


Aedes albopictus female mosquito. This mosquito is taking a blood meal from a human host.[2].

Aedes albopictus

Aedes albopictus (Ae. albopictus) mosquitoes also have a dark color with white-banded legs, but with a longitudinal white stripe along its head and thorax [17]. Ae. albopictus mosquitoes are most active during the dawn hours and the afternoon [17]. Unlike Ae.ae mosquitoes, Ae. albopictus females are aggressive day biters [17]. They prefer biting any exposed skin on humans, but they also take blood meals from both domestic and wild mammals [16]. Ae. albopictus mosquitoes bite rapidly, allowing to take a blood meal before possibly being swatted [16]. About 4-5 days after a blood meal, female Ae. albopictus are ready to lay eggs in natural or artificial containers with water [16]. Like Ae.ae mosquitoes, Rainfall allows larvae to hatch and it takes 7-9 days for larvae to reach adulthood [16]. On the other hand, adult Ae. albopictus eggs persist through the winter in temperate temperature zones, while adult mosquitoes can live year-round in tropical and subtropical regions [16]. Aedes albopictus mosquitoes are also primary vectors of Dengue virus and Chikungunya virus, but they have also been found to transmit Japanese encephalitis virus, yellow fever, and Heartworm parasites [20].




Viruses vectored by Aedes Mosquitoes

Dengue virus (DENV)

Dengue Fever Poster from onlineAP, a news journal in India [3].

Dengue Virus (DENV), a Flavivirus of the family Flaviridae, is a positive sense RNA virus. DENV is vectored by Aedes mosquitoes [20]. Aedes Mosquitoes become infected with DENV when uninfected females take a blood-meal from infected hosts [7]. From there the virus replicates within the mosquito and is translocated to the mosquito salivary gland in order to be transmitted upon the next blood-meal [7]. DENV has 5 serotypes, or distinct genotypes, with DENV-2 being the most aggressive. DENV is the cause of Dengue Fever, a mild fever in most cases, but also a serious Hemorrhagic fever (DHF) that can cause liver failure and death [18]. DHF occurs most often in the elderly and early youth populations [18]. DENV infects 100,000,000 million people a year worldwide, causing serious cases of DHF in 500,000 people, and 20,000 deaths [20]. Currently there is no cure or vaccine to treat DENV, although DENV is found in tropical and subtropical regions, and over 1 billion people reside in the range of this virus [20]. Currently Dengue Virus is in Hawaii, spreading from two confirmed cases in October 2015 to over one hundred cases as of November 2015.


Chikungunya rash on the right foot of a victims.CDC.
Cryo-electron microscopy reconstruction of Chikungunya virus. From EMDB entry 5577.[4]

Chikungunya virus (CHIKV)

Chikungunya Virus (CHIKV), an Alphavirus of the family Togaviridae, is also a positive sense RNA virus [14]. Aedes aegypti mosquitoes were thought to be the sole vector of CHIKV [5]. However, it was discovered that Aedes albopictus mosquitoes can also transmit CHIKV [5]. (Lamballerie et al, 2008) saw that CHIKV virus infecting Aedes albopictus mosquitoes had an Alanine at amino acid 226 on the E1 gene mutated to a valine [5]. Through a single amino acid mutation in the virus, Aedes albopictus mosquitoes acquired the ability to vector and transmit Chikungunya virus (CHIKV) [5]. CHIKV is the cause of mild fever accompanied with an incapacitating arthralgia, or joint pain that can persist for months or even years [11]. Upon the bite of an infected Aedes mosquito, CHIKV replicates within dermal layer of skin and disseminates in to the blood stream [10]. From here, CHIKV predominantly infects the muscles, joints, skin, but also the liver, spleen, and brain, to some capacity [10]. Host immunity launches an inflammatory cell response which leads to the pain and inflammation experienced with this virus [10]. There is also no cure, or vaccine for this virus, and treatments are based on patient symptoms [19]. In 2013, the first case of CHIKV reached the Americas in the Caribbean [19]. According to the CDC, before 2013, there were 28 cases of CHIKV infection a year in the US. By the end of 2014 there were 2,811 in the U.S, and 4,710 in U.S. territories.


Wolbachia

What is Wolbachia

Transmission electron micrograph of Wolbachia within an insect cell. Photo was originally taken by Scott O'Neill from the paper Genome Sequence of the Intracellular Bacterium Wolbachia. PLoS Biol 2/3/2004: e76. doi:10.1371/journal.pbio.0020076 , and edited by eliminate dengue.com [5]

Wolbachia pipentis is a maternally inherited, Gram-negative bacterium [13]. It was discovered within a Culex pipentis mosquito in 1924 by Michael Hertig and Samuel Wolbach [13]. Wolbachia works as an endosymbiont bacterium that takes advantage of its host reproduction in order to proliferate [13]. Wolbachia induces Cytoplasmic incompatibility (CI) in its host [2]. CI is the failure of a sperm and egg to produce an embryo that supports viable offspring [2]. Wolbachia can induce CI both uni-directionally and bi-directionally. Unidirectional CI occurs when a Wolbachia-infected male attempts to mate with an uninfected female male leading to the death of the offspring in the embryo. Bi-directional CI occurs in strain-specific manner, where females and males infected with different strains of Wolbachia can’t form viable offspring [2]. Although the mechanism isn’t well known, Wolbachia are believed to alter chromosomes within their female host. This affect causes complications within mitotic divisions that obstruct the synchrony between sperm and egg [7]. Wolbachia is found within 40% of all terrestrial arthropods. With that said, there are an abundance of Wolbachia strains that are named by the host from which they were isolated.

Diagram depicting the process of Cytoplasmic Incompatibility (CI).[6]

Wolbachia Controls Mosquitoes and Viruses

Within the last 10-15 years, Wolbachia has been tested against mosquitoes and virus, as an agent to control the spread of mosquito-borne arboviruses [1-6]. Wolbachia has been shown to be an effective treatment against viral proliferation in mosquitoes, viral transmission from mosquitoes, and even the ability of the mosquito to be infected [3]. Wolbachia has been shown to either inhibit or reduce levels of DENV, CHIKV, WNV, YFV, Plasmodium (cause of malaria) in multiple mosquito species including Aedes mosquitoes [3]. However Wolbachiahas shown cryptic results that raise trivial questions about the activity of Wolbachia within mosquito vectors. Wolbachiahas demonstrated repressive behavior against virus, but at evolutionary cost, or even the enhancement of viral transmission [23]. Also mosquito life span was seen to increase when Wolbachiaengaged in antiviral response [1].


Wolbachia controls Mosquito and virus inability control

Mosquito Vector Control

Wolbachia has been shown to shorten the life span of Aedes aegypti mosquitoes [7]. In 2009, McMeniman and group, infected Aedes aegypti mosquitoes with Wolbachia strain w(mel)pop for 3 years creating a lineage of Wolbachia infected mosquitoes [7]. They then compared their survival percentage to an Aedes aegypti line uninfected with Wolbachia . They found that both male and female mosquitoes infected with the wMel pop strain of Wolbachia lived (20-40 days) less than uninfected mosquitoes [7]. In addition, mosquitoes lived half the life span of uninfected mosquitoes in conditions modeling the environment. In addition Wolbachia infection induced (CI) strongly among the infected mosquito strain [7]. Inheritance of Wolbachia within Female mosquitoes, the determinant of successful CI, was not statistically different from 99% among this population [7]. This ensured that the Wolbachia infection had the ability to persist in wild Aedes aegypti populations [7]. Female mosquitoes infected with a pathogen must live about 16 days, in order for the pathogen to mature for transmission [7]. By shortening the life span of adult mosquitoes their chances of acquiring a viral infection were lowered along with their chances of transmitting the virus to another host.

Antiviral Response Mechanisms

Density Dependence

One proposed mechanism for Wolbachia’s antiviral response is characterized by the density of Wolbachia in a cell. In other words, density dependence is the amount of Wolbachia distributed within a cell or tissue compared to that of the virus within a host mosquito cell. Within the wMel strain, high virus reduction was seen in cells with high Wolbachia density. This finding was consistent with multiple studies within the field [1, 2, 3,]. However no direct mechanism for the density dependent antiviral response of Wolbachia was uncovered. The density dependent mechanism was refuted in a study by Lu and colleagues in 2012 [6]. Lu infected Aedes albopictusmosquito cells and Aedes aegypti mosquitoes with Wolbachia and then subjected them both to virus for an extended period of time. Lu found that Wolbachia reduced DENV titer within the mosquito [6]. Lu also witnessed that Wolbachia<did induce some density dependent inhibition of DENV in mosquito cells [6]. Lu saw a negative correlation between Wolbachia densities within cells compared to virus density [6]. However Lu measured the amount of Wolbachia within Ae.ae midgut and Ae. albopictus cells, and determined that the amount of Wolbachia present was not enough to inhibit DENV infection [6]. According to the model, Lu estimated that the amount of Wolbachia necessary to inhibit DENV infection was at least 99.5% more than what was found in the either the mosquito midgut or in cells [6]. Therefore Lu concluded that other antiviral mechanisms may be effected by Wolbachia density like, immune response, competition, or a direct antiviral response from Wolbachia. Lu proposed that these mechanisms were the root of Wolbachia’s antiviral response and became enhance with high Wolbachia density [6]. Lu measured the amount of a Toll pathway protein, DEFD, expressed in the presence of Wolbachia. Lu found that an increase in Wolbachia density also yielded an increase in the expression of DEFD [6]. With this information Lu concluded that density of Wolbachia within a cell does play a part in the inhibition of a viral infection, but it isn’t the only mechanism at work [6].

Competition for Host resources

Wolbachia is thought to cause reduced virus titer by competing with the virus for host mechanisms and materials. Wolbachia is known to compete with its mosquito host for essential nutrients [24]. Caragata fed Wolbachia-infected Aedes aegypti mosquitos’ non-human blood in order to assess fitness changes under these starved conditions. Caragata examined the changes in Cholesterol and amino acids as they play a major role in mosquito egg development. Caragata found that levels of Cholesterol and amino acid levels decreased as a result of competition between Wolbachia and its host mosquito [24]. It is known that amino acids are necessary for some viruses to reproduce in cells. CHIKV was unable to grow in stable mosquito cells without the presence of cysteine in the media [9]. DENV was not able to grow in cells without the presence of glycine. Therefore, based off of these observations, when a mosquito is infected with a virus, it is thought that Wolbachia and the virus are competing for these resources in the host. In other words, Wolbachia may be able to reduce virus infection by limiting the amount of host resources available for viral proliferation. Cholesterol can inhibit DENV from entering the cell, and also complicates its development in the early stages of infection [8]. In this way, Wolbachia may induce fitness changes in its host by leaving them more susceptible to virus infection. However, based off this evidence, a trade off may exist. As Wolbachia consumes resources for itself, it may also be inadvertently inviting the introduction of competition from viruses.

Immune Function

Wolbachia has been shown to activate the immune system of host mosquitoes by causing the upregulation of immune response genes [1, 2, 4, and 6]. Depending on the organism, genes belonging to the Toll pathway or related pathway are upregulated.

ROS Toll pathway Activiation

The Toll pathway is an immune pathway activated by Toll-like receptors (TLR). Toll-like receptors are proteins that recognize microbes and activate the innate immune system. Bian and colleagues investigated if Wolbachia suppressed DENV in Aedes aegypti mosquitoes [1] (Bian et al., 2010). After Bian infected Wolbachia-infected and uninfected Ae.ae mosquitoes with DENV, he compared the amount of DENV present within the head and thorax of each population, assessed the amount of expression of immune cells induced by each population, and tested if either population caused an increase in Ae.ae longevity [1]. Bian found that the presence of Wolbachia lowered dissemination of DENV from the head to the thorax [1]. In addition he found that Wolbachia effectively increased the life span of these infected mosquitoes. Bian also noticed an increase in the upregulation of defensins, cecropin, REL1, SPZ1A, and GNBPB1, known to be a part of the Toll pathway in mosquitoes [1]. This suggested that Wolbachia activated the Toll pathway of host mosquitoes, which would heighten immune response to presence of pathogens. This in turn would lead to a decrease in the prevalence of pathogens. Pan and colleagues, including Bian, set out to find a possible mechanism for how Wolbachia activated the Toll pathway [25]. Pan used microarrays on 7-day old Wolbachia-infected mosquito midgut to assess gene transcripts [25]. Pan found that most transcripts were based in immunity and oxidative stress. In an assay to analyze gene expression, REL1 and GNBPB1 were both up regulated [25]. Therefore they saw Wolbachia induced the Toll pathway [25]. Pan went on to find that the presence in Wolbachia also induces oxidative stress within the cell by producing Reactive oxygen species (ROS) [25]. Pan treated Ae.ae mosquitoes with Hydrogen peroxide in order to see if Reactive oxygen species could induce the Toll pathway [25]. Pan found that Toll pathway genes were upregulated in these conditions, confirming the activation of the Toll pathway [25]. Pan then determined that Wolbachia-induced ROS was the cause of the activation of the Toll pathway by silencing Wolbachia oxidases and looking for a response in the Toll pathway [25]. Pan found that the Toll pathway was also silenced as a result of the silencing of Wolbachia’s oxidases [25]. Pan then determined that the Toll pathway was necessary for antioxidant expression in these mosquitoes [25]. Therefore Pan uncovered that a mechanism for Wolbachia’s ability to activate the Toll pathway in Aedes aegypti, was through Wolbachia’s production of ROS [25].

Gene Expression Regulation

Wolbachia- mediated immune activation was investigated in Anopheles gambiae, another important mosquito vector for malaria. This mosquito is known to have an immune activation pathway related to the Toll pathway [4]. Kambris and colleagues found upregulation of genes known to have an inhibitory effect on Plasmodium, the cause of malaria [4]. However it seems as though the Immune Deficiency pathway (IMD), effects Wolbachia density, and leads to a decrease in Wolbachia’s pathogenicity [4].


Effects of Wolbachia’s strain specific diversity on mosquito vectorship

Wolbachia-mediated antiviral protection against diverse RNA viruses in Drosophila and mosquito hosts. This table is adapted to show the effects of various Wolbachia strains on dangerous human pathogens in multiple insect hosts. This table was made by Johnson KN, in the study Johnson KN. Bacteria and antiviral immunity in insects. . 2015;8: 97-103.[7]

Connection to Healthcare

Wolbachia- mediated vector control represents a promising way to prevent the spread of multiple deadly arboviruses. As stated earlier, Dengue virus and Chikungunya virus have no vaccines or cure, but are only treated through patient symptoms. At the same time, these viruses continue to spread throughout the globe annually, virtually unchecked by medicine. As the field works toward a specific vaccine or cure, more individuals continue to contract infections, suffer from symptoms, and possibly die from that same virus or another. By interrupting the vectorship between mosquitoes and vector-borne arboviruses, we attack a common ground between virus types and mosquito species. Wolbachia seems to act as a universal key to an issue with a lot variation, in its ability to block many types of viruses within many mosquitoes.

References

[1]Bian G, Xu Y, Lu P, Xie Y, Xi Z. The Endosymbiotic Bacterium Wolbachia Induces Resistance to Dengue Virus in Aedes aegypti. . 2010;6: e1000833.

[2]Blagrove MS, Arias-Goeta C, Failloux AB, Sinkins SP. Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus. Proc Natl Acad Sci U S A. 2012;109: 255-260.

[3]Johnson KN. Bacteria and antiviral immunity in insects. . 2015;8: 97-103.

[4]Kambris Z, Blagborough AM, Pinto SB, Blagrove MSC, Godfray HCJ, Sinden RE, et al. Wolbachia Stimulates Immune Gene Expression and Inhibits Plasmodium Development in Anopheles gambiae. . 2010;6: e1001143.

[5]de Lamballerie X, Leroy E, Charrel RN, Ttsetsarkin K, Higgs S, Gould EA. Chikungunya virus 33s adapts to tiger mosquito via evolutionary convergence: a sign of things to come? . 2008;5: 33.

[6]Lu P, Bian G, Pan X, Xi Z. Wolbachia Induces Density-Dependent Inhibition to Dengue Virus in Mosquito Cells. . 2012;6: e1754.

[7]McMeniman CJ, Lane RV, Cass BN, Fong AW, Sidhu M, Wang YF, et al. Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti. Science. 2009;323: 141-144.

[8]Lee CJ, Lin HR, Liao CL, Lin YL. Cholesterol effectively blocks entry of flavivirus. J Virol. 2008;82: 6470-6480.

[9]Sasao F, Igarashi A, Fukai K. Amino acid requirements for the growth of Aedes albopictus Clone C6/36 cells and for the production of dengue and chikungunya viruses in the infected cells. Microbiol Immunol. 1980;24: 915-924.

[10]Lo Presti A, Lai A, Cella E, Zehender G, Ciccozzi M. Chikungunya virus, epidemiology, clinics and phylogenesis: A review. . 2014;7: 925-932.

[11]Couderc T, Lecuit M. Chikungunya virus pathogenesis: From bedside to bench. Antiviral Res. 2015;121: 120-31.

[12]Tsetsarkin KA, Vanlandingham DL, Mcgee CE, Higgs S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. . 2007;3: 1895-1906.

[13]Kozek W, Rao R. The discovery of Wolbachia in arthropods and nematodes–A historical perspective. . 2007.

[14]Centers for Disease Control and Prevention. (2010). Symptoms of Chikungunya Fever.

[15]Centers for Disease Control and Prevention. (2012). Aedes aegypti Fact Sheet.

[16]Centers for Disease Control and Prevention. (2012). Aedes albopictus Fact Sheet.

[17]Centers for Disease Control and Prevention. (2012). Comparison of Dengue Vectors.

[18]World Health Organization. (2015). Dengue and severe dengue.

[19]World Health Organization. (2015).Chikungunya.

[20]World Health Organization. (2015).Dengue control: The mosquito.

[21]World Health Organization. (2014). Vector-borne diseases

[22]R, Hammerstein P. Still a Host of Hosts for Wolbachia: Analysis of Recent Data Suggests That 40% of Terrestrial Arthropod Species Are Infected. . 2012;7: e38544.

[23]M, Marini F, Desiderio A, Puggioli A, Moretti R. Wolbachia density and cytoplasmic incompatibility in Aedes albopictus: concerns with using artificial Wolbachia infection as a vector suppression tool. PLoS One. 2015;10: e0121813.

[24]Caragata EP, Rances E, O'Neill SL, McGraw EA. Competition for Amino Acids Between Wolbachia and the Mosquito Host, Aedes aegypti. Microb Ecol. 2014;67: 205-218.

[25]X, Zhou G, Wu J, Bian G, Lu P, Raikhel AS, et al. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Proc Natl Acad Sci U S A. 2012;109: E23