The role of environmental, virological and vector interactions in dictating biological transmission of arthropod-borne viruses by mosquitoes.

CHAPTER TWO

The Role of Environmental, Virological and Vector Interactions in Dictating Biological Transmission of Arthropod-Borne Viruses by Mosquitoes Joan L. Kenney, Aaron C. Brault1 Arbovirus Research Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, U.S. Centers for Disease Control and Prevention, Fort Collins, Colorado, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Background 2. Vectorial Capacity 3. Oral Vector Infection 3.1 Viral determinants of infection 3.2 Receptor-mediated midgut infection 3.3 Vector genetics that modulate viral infection 3.4 Blood-feeding factors and vector infection 4. Midgut Escape and Dissemination 4.1 Intrahost viral populations 4.2 Viral population bottlenecks 4.3 Physiological and pathological changes imparted by arboviral infection 5. Environmental Variables 6. Mosquito-Specific Viruses 6.1 Superinfection exclusion 6.2 Vertical transmission of arboviruses in mosquitoes 7. Utilizing Mosquito Biology to Inhibit Arbovirus Infection 7.1 Mosquito innate immune response 7.2 Microbiota 8. Conclusions Acknowledgments References

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Abstract Arthropod-borne viruses (arboviruses) are transmitted between vertebrate hosts and arthropod vectors. An inherently complex interaction among virus, vector, and the Advances in Virus Research, Volume 89 ISSN 0065-3527 http://dx.doi.org/10.1016/B978-0-12-800172-1.00002-1

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2014 Elsevier Inc. All rights reserved.

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Joan L. Kenney and Aaron C. Brault

environment determines successful transmission of the virus. Once believed to be “flying syringes,” recent advances in the field have demonstrated that mosquito genetics, microbiota, salivary components, and mosquito innate immune responses all play important roles in modulating arbovirus transmissibility. The literature on the interaction among virus, mosquito, and environment has expanded dramatically in the preceding decade and the utilization of next-generation sequencing and transgenic vector methodologies assuredly will increase the pace of knowledge acquisition in this field. This chapter outlines the interplay among the three factors in both direct physical and biochemical manners as well as indirectly through superinfection barriers and altered induction of innate immune responses in mosquito vectors. The culmination of the aforementioned interactions and the arms race between the mosquito innate immune response and the capacity of arboviruses to antagonize such a response ultimately results in the subjugation of mosquito cells for viral replication and subsequent transmission.

1. BACKGROUND Arthropod-borne viruses (arboviruses) are grouped by their common means of transmission to vertebrate hosts by the bite of infected arthropod vectors. Although arboviruses have been documented to be transmitted between vertebrate hosts by flies, sandflies, midges (Depaquit, Grandadam, Fouque, Andry, & Peyrefitte, 2010; Kramer, Jones, Holbrook, Walton, & Calisher, 1990; Mellor, Boorman, & Baylis, 2000), cliff swallow bugs (Brown, Moore, Young, Padhi, & Komar, 2009), and ticks (Nuttall, Jones, Labuda, & Kaufman, 1994), the majority are transmitted by mosquitoes and therefore mosquito-borne viruses will be the focus of this chapter. Typically, these viruses exist in a dual-host cycle between the mosquito and some vertebrate host such as a bird, rodent, amphibian, or primate. Infection of mosquitoes with arboviruses occurs in a dose-dependent manner (Weaver, 1994) following ingestion of an infectious blood meal and thus only vertebrate hosts that manifest sufficient titers can contribute to the transmission cycle. Mosquito-borne arboviruses belong to a number of families including Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, and Rhabdoviridae. With few exceptions, such as dengue viruses (DENV) 1–4, yellow fever virus (YFV), and chikungunya virus (CHIKV), humans serve as “dead-end” hosts by not manifesting sufficient viremias for the oral infection of additional vectors to propagate the viral cycle. In addition to dualhost (vertebrate and vector-infecting viruses) mosquito-borne viruses, a number of mosquito-specific viruses have been identified for which the

Arboviral Interactions with Mosquitoes

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capacity for replication in vertebrate cells has not been observed. These viruses have been described extensively in the family Flaviviridae (Cook et al., 2012) and recently in the families Togaviridae (Nasar et al., 2012) and Bunyaviridae (Marklewitz et al., 2013). The discovery of these viruses should allow for in-depth study of mechanisms of vertical transmission of different viral families in mosquitoes as well as novel mechanisms of RNAi antagonism of vector species in addition to the fundamental elements that restrict host range of these viruses. Arboviruses can be transmitted to a vertebrate host via a mosquito vector by two distinct mechanisms: mechanical or biological transmission (Hardy, Houk, Kramer, & Reeves, 1983). Mechanical transmission occurs by direct contact of contaminated mouthparts of the arthropod vector with the vertebrate host, thus not requiring amplification of the virus within the vector (Gray & Banerjee, 1999; Kaufman & Nuttall, 1996; Mayr, 1983). Biological transmission, in contrast, necessitates the direct amplification of the virus in mosquito tissue prior to transmission. As such, amplification of the virus in mosquito cells has resulted in a number of evolutionary processes that will be addressed throughout this chapter for the virus to directly antagonize the innate immune response of the mosquito as well as offset indirect fitness effects on viral replicative homeostasis. Nevertheless, viruses such as West Nile virus (WNV) for which biological transmission is the predominant mechanism for transmission by mosquitoes have been documented to be mechanically transmitted by stable flies through contaminated mouthparts (Doyle et al., 2011; Johnson, Panella, Hale, & Komar, 2010). A series of intrinsic and extrinsic factors such as the ability to productively infect the midgut epithelium of the vector combine to determine the efficacy of a virus–vector relationship (Chamberlain & Sudia, 1961). Several examples will be provided for various arboviruses to demonstrate these barriers throughout this chapter. Biological transmission of an arbovirus in a mosquito vector entails passing through a number of physical and physiological barriers in order for the virus to be imbibed by a mosquito in an infectious blood meal and transmitted upon expectoration during probing and feeding of the mosquito at the initiation of the subsequent gonotrophic cycle (Fig. 2.2). Infection of midgut epithelial cells (Fig. 2.2, panel 1a and b), productive viral propagation, dissemination of virus from midgut epithelial cells to cell populations present in the hemocoel, infection of salivary glandular acinar cells (Fig. 2.2, panel 2a), and deposition of virus in the apical cavities and salivary ducts of the salivary gland for transmission (Fig. 2.2, panel 2b) during feeding are required to

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complete the cycle. The time period between the ingestion of an infectious blood meal and the transmission of an arbovirus is known as the extrinsic incubation period (EIP) as this is the period observed in which the arbovirus was not replicating in the vertebrate (or intrinsic) host. Given the multiple sets of intricate physical and evolutionarily selective barriers that have arisen for the establishment of persistent infections of arboviruses in mosquito vectors and the potential that infection of mosquitoes with an arbovirus has been documented to occur in the absence of actual amplification in the vertebrate host (Higgs, Schneider, Vanlandingham, Klingler, & Gould, 2005; Reisen, Fang, & Martinez, 2007), it is becoming clear that such a simple designation as “extrinsic incubation” in the invertebrate host might be an improper descriptive term for these complex interactions. As previously alluded to, viral, vector, and environmental factors of both an intrinsic and extrinsic nature can alter each stage of biological transmission. A number of barriers to infection have been described for arthropod-borne viruses. These include (1) barrier to midgut infection (midgut infection barrier) that results in the failure of a virus to bind, enter, and/or replicate within the midgut epithelial cells (typically associated with the presence of receptors on the surface of the midgut epithelial cells); (2) a barrier to dissemination (midgut escape barrier) from productively infected midgut epithelial cells; (3) a barrier to the productive infection of acinar cells of the salivary glands; and finally (4) a barrier to the replication within and escape from the salivary gland cells (Hardy et al., 1983). The original description of these barriers focused on the environmental (temperature) that restricted or promoted viral replication in the mosquito and physical barriers that were found between these replication sites present on the midgut epithelial cells such as receptors, the thickness and pore size limitations of the basal lamina (BL) that could prevent viral dissemination from infected midgut epithelial cells, and similar barriers for infection and release from the acinar cells of the salivary glands. The recent advances in understanding of the complex innate immune responses that mosquitoes can mount to arboviruses has indicated that the barriers are also significantly affected by either direct innate immune responses targeting viral replication intermediates or indirect effects from microbiome priming of the mosquito innate immune system (Ramirez et al., 2012; Xi, Ramirez, & Dimopoulos, 2008).

2. VECTORIAL CAPACITY Vectorial capacity is described as the “combined effect of all of the physiological, ecological, and environmental factors relating vector, host, and


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Figure 2.1 Diagram depicting the interaction between the vector, virus, and environmental factors. Area in the center of the diagram depicts concordance of all three of these factors that would hypothetically lead to enhanced interactions for optimal transmissibility of arboviral agents.

pathogen that determine the ability of a given mosquito species to serve as a competent vector for a particular virus” (Garrett-Jones, 1964; Hardy et al., 1983). This combined effect is stylized as the overlapping components of mosquito, virus, and environmental variables in Fig. 2.1; brown centroid composite of all three variables. The concept of vectoral capacity includes factors other than those associated with direct biological interplay between virus and mosquito. Myriad factors can strongly influence vectorial capacity such as mosquito longevity (inclusive of vector-intrinsic factors not directly associated with viral infection) and blood-feeding preference to name a few that directly facilitate the necessary interplay between these elements. This chapter will focus on intrinsic factors that dictate the interplay of these variables and their resulting effect on the vectorial capacity of a virus–vector relationship. Previous studies utilizing intrathoracic inoculation of multiple mosquito vectors not susceptible to oral infection have demonstrated that the ability to

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infect the midgut epithelium is a critical barrier for dictating the capacity of a mosquito to become infected with a particular arbovirus (Hardy et al., 1983; McLean, 1955; Merril & TenBroeck, 1935). Poorly competent mosquito vectors have been demonstrated to serve as potential vectors during arboviral outbreaks due to the overriding importance of other variables such as mosquito density (Miller, Monath, Tabachnick, & Ezike, 1989) and/or elevated host titers (Komar, 2003; Turell et al., 2005). Seasonal variations in the susceptibility of mosquitoes to infection can occur due to environmental factors (see Hardy & Reeves, 1990; Reisen, Fang, & Martinez, 2006 for marked changes in ID50 with season; also Meyer, Hardy, Presser, & Bruen, 1983 for the effect of parity) and mosquito genetics (Reisen, Barker, Fang, & Martinez, 2008) and must be considered when assessing the vector competency of field-populations. The fundamental environmental (Reisen, Hardy, & Presser, 1997) and mosquito genetic or epigenetic basis for these observed alterations in susceptibility has not been established; however, differences in the seasonal microbiota of mosquitoes that stimulate indirect innate effector genes or possibly direct inhibition due to seasonal infection prevalence differences with heterologous arboviral agents could contribute to such variability. Each of these factors will be discussed specifically in subsequent sections.

3. ORAL VECTOR INFECTION Biological transmission initially necessitates infection and replication of the virus in the midgut epithelial cells. A number of critical gaps in knowledge regarding the epithelium of mosquitoes as it pertains to arboviral susceptibility exist that handicap a thorough assessment of oral infection of mosquito vectors. For instance, morphological and physiological characterization of the uniformity and impermeability of epithelial cell types and epithelial integrity throughout the midgut have not been well established (Houk & Hardy, 1979). As a result, the importance of different epithelial cells and cellular markers dictating differential susceptibility to arboviral infection of these cells has also not been systematically undertaken. Oral susceptibility to differential arboviral infection has been studied extensively in the context of various virus and mosquito vector models (Hardy, Reeves, & Sjogren, 1976; Houk, Arcus, Hardy, & Kramer, 1990; Scott, Hildreth, & Beaty, 1984; Weaver, Scott, Lorenz, Lerdthusnee, & Romoser, 1988). The oral route of infection has been examined most thoroughly as infection of the midgut epithelium serves


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Figure 2.2 Diagram of mosquito anatomy designating the different barriers to infection, dissemination, and transmission. Panel 1: midgut epithelium (a) designation of virus moving from apical to basolateral side of epithelial cell; (b) sagittal section of the gut demonstrating the clotting and movement of virions to the periphery adjacent to the midgut apical villi. Panel 2: salivary gland infection (a) lateral view demonstrating viral particle exposure of salivary gland acinar cells from the basal side (b) close-up demonstrating passage of the virus from the basal to the apical egress of the salivary gland epithelia cells into the salivary gland duct. Panel 3: depiction of viral infection of ovarian tissue for potential vertical transmission. Abbreviations: DD, dorsal diverticulum; HC, hemocoel; MT, Malpighian tubules; VD, ventral diverticulum.

as the initial barrier determining vector competence (hereto the term “vector competence” refers to the overall capacity of a mosquito to become orally infected and transmit an arbovirus). For a mosquito to be a competent vector for an arbovirus, the ingested viral particles must infect the epithelial cells of the midgut (Fig. 2.2, panel 1a and b) in a receptormediated fashion at the apical surface and subsequently replicate within the cell. A number of physiological changes also occur in the gut following blood feeding that can alter the receptivity of the mosquito midgut to viral infection such as exposure of the virions to trypsin and chymotrypsin, which are released into the lumen of the gut as part of the digestive process for the mosquito to metabolize the blood meal. Use of trypsin inhibitors, for example, has been shown to reduce the oral susceptibility

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of Aedes aegypti to DENV-2 infection and dissemination (Molina-Cruz et al., 2005). Additionally, transcriptome studies have demonstrated that the miRNA profile of several mosquitoes show distinctive changes following blood feeding (Hussain, Walker, O’Neill, & Asgari, 2013). Recent studies have demonstrated that arboviruses can encode microRNA (miRNA) target sequences in their 30 UTR that subjugate the host’s posttranscriptional gene regulatory pathways in order to restrict tissue tropism and alter vertebrate pathology (Trobaugh et al., 2013). Similarly, miRNAs have been described to be encoded in the 30 UTR of WNV which targets mosquito GATA4 mRNA expression and is positively associated with WNV replication in cultured mosquito cells (Hussain et al., 2012). Furthermore, miRNA expression patterns can be modified directly as a result of infection with arboviruses, indicating that the viruses can directly manipulate the cellular environment, altering mosquito cellular receptiveness for replication. For instance, DENV-2 infection of Ae. aegypti demonstrated modulations in up to 32 different miRNAs, many of which have been implicated with host transcriptional regulatory and signal transduction patterns known to be involved in viral replication and dissemination (Campbell, Harrison, Hess, & Ebel, 2014). The effects of such miRNA expression patterns on receptiveness and refractoriness to different arboviral infections remains to be seen. Differential infection thresholds of mosquito vectors have historically been performed with closely related viruses for the implication of virally encoded determinants of altered vector competence (Brault et al., 2004; Brault, Powers, & Weaver, 2002; Kramer & Scherer, 1976; Woodward, Miller, Beaty, Trent, & Roehrig, 1991). Evidence exists indicating that both viral, vector, as well as extrinsic factors such as larval nutrition and temperature can affect midgut infection of mosquitoes with different viruses (Day & Van Handel, 1986; Grimstad & Haramis, 1984; Kramer, Hardy, & Presser, 1983; Reeves, Hardy, Reisen, & Milby, 1994; Turell, 1993; Turell & Lundstrom, 1990; Turell, Rossi, & Bailey, 1985). A number of hypotheses have been proposed to explain the resistance of a mosquito to infection with particular viruses, including (1) diversion of the ingested blood meal into the ventral diverticulum; (2) peritrophic membrane filtration of viruses; (3) digestive inactivation of virions while within the midgut lumen; (4) cellular charge/charge distribution differences within the mesenteronal epithelia; and (5) differential expression of specific receptors on the apical surface of the epithelia of infection susceptible mosquitoes (Hardy et al., 1983).


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Virus must be taken in an infectious blood meal from a viremic host in suitable quantity to infect the mesenteronal epithelium. Diversion of virus into the ventral diverticulum (Fig. 2.2) as opposed to the midgut, where infection of the epithelia occurs, has been used as a possible explanation for differences in viral infection. However, there is no evidence that different viruses are alternatively shuttled to the diverticulum. In fact, only blood meals containing sucrose contents in excess of 2.5% have been determined to be routed to the diverticulum with regularity (Hosoi, 1954). Although the pore size of the peritrophic membrane has been determined to be smaller (20–30 nm) than the smallest known arboviruses (Houk, Obie, & Hardy, 1979; Richards & Richards, 1977), the formation of the peritrophic membrane occurs much more slowly than infection of the mesenteron (Richards & Richards, 1977). Similarly, maximal secretion of the digestive enzymes, trypsin and chymotrypsin, within the lumen of Culex tarsalis mosquitoes has been measured to occur between 12 and 16 h postblood feeding. Conversely, experiments exposing LaCrosse virus (LACV) to digestive enzymes, potentially found in the midgut lumen, have resulted in cleavage of the G1 and G2 glycoproteins to allow for more efficient binding to mosquito cells (Ludwig, Christensen, Yuill, & Schultz, 1989; Ludwig, Israel, Christensen, Yuill, & Schultz, 1991).

3.1. Viral determinants of infection A number of studies have focused on identifying particular viral genetic determinants that could be driving successful infection of mosquitoes as hosts. In some cases, the vital regions required for infection are few in number and easily identified. For instance, following the La Re´union island outbreak of the alphavirus CHIKV in 2005 and 2006, a single mutation in the E1 glycoprotein at position 226 was shown to afford the virus significantly enhanced ability to infect and disseminate within Aedes albopictus, but not for the original primary vector Ae. aegypti (Tsetsarkin, Vanlandingham, McGee, & Higgs, 2007). In 2009, a secondary corollary mutation in the CHIKV outbreak strain was observed at position 210 of the E2 glycoprotein and shown to affect the ability of the virus to infect Ae. albopictus midgut cells (Tsetsarkin & Weaver, 2011). Mutations in the positions 55 and 70 of the E2 glycoprotein for Sindbis virus (SINV) were also determined to culminate in enhanced midgut infectivity for Ae. aegypti (Pierro, Powers, & Olson, 2007). Studies with western equine encephalitis virus (WEEV) support the role of a single mutation at E2 218 having deleterious effects on vector

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infection; however, the reciprocal mutation introduction did not confer mosquito infectivity to a mouse-adapted WEEV strain (Mossel et al., 2013). The trend continues with Venezuelan equine encephalitis (VEEV) where a single mutation at position 218 of the E2 glycoprotein was characterized and appears to permit the enzootic VEEV strains to infect, replicate, and be transmitted by the exclusive epizootic mosquito vector, Aedes taeniorhynchus (Brault et al., 2004, 2002). Interestingly, determinants for infection of the enzootic VEEV mosquito vector do not appear to be limited to the E2 glycoprotein and likely also involve portions of both the structural and nonstructural protein genes (Kenney, Adams, Gorchakov, Leal, & Weaver, 2012). A number of flavivirus chimeric virus studies have indicated that regions outside the prME envelope genes modulate infection of mosquito cells in vitro (Charlier et al., 2010; Johnson et al., 2003, 2004; Pletnev & Men, 1998) and in in vitro mosquito models (Brault et al., 2011; Hanley et al., 2005). However, some chimeric flavivirus studies also implicate structural regions as contributing determinants for infection of the mosquito vector (Engel et al., 2011; Pletnev, Bray, Huggins, & Lai, 1992). In vitro experiments in C6/36 cells with prME chimeras between WNV and St. Louis encephalitis virus (SLEV) suggest that portions of both the nonstructural and structural gene regions are determinants for vector infection; however, the same chimeric viruses compared in Cx. tarsalis cells indicated that structural genes were the primary determinants (Maharaj et al., 2012). Although the data are limited, there appears to be a trend demonstrating that viruses circulating predominantly in enzootic transmission cycles, such as enzootic VEEV and SLEV and their respective vectors, appear to have more than one region of genome that modulates successful replication and dissemination. This could be a result of a long history of coadaptation between the vector and virus that has resulted in a more stable interaction. For instance, both WNV and epizootic strains of VEEV require approximately a minimum of 100-fold higher titers to efficiently infect their respective vectors at a high enough incidence to maintain the transmission cycle. However, the vectors for both enzootic VEEV and SLEV have a lower threshold of infection, suggesting a more enduring commensalism between the vector and virus in an enzootic cycle. While the majority of these studies have been conducted using reverse genetic systems of flaviviruses and alphaviruses, some recent work with bunyaviruses has also been performed despite their negative strand, segmented genome. Using a recombinant Rift Valley fever system (Bird et al., 2008; Bird, Albarino, & Nichol, 2007; Gerrard, Bird, Albarino, & Nichol,


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2007), Crabtree et al. were able to demonstrate that deletions of the individual nonstructural proteins from the small and medium segments (NSs and NSm) had a deleterious effect on virus infection and dissemination within Ae. aegypti and Culex quinquefasciatus. The combination of deletions resulted in the highest attenuation phenotype and ablated infection in Ae. aegypti (Crabtree et al., 2012).

3.2. Receptor-mediated midgut infection To date, it has been suggested that arboviruses, particularly alphaviruses and flaviviruses, invade the host cell through receptor-mediated endocytosis with a requirement for an acidic change in pH (Chu, Leong, & Ng, 2005; Helenius & Marsh, 1982; Kielian, 1995; Kielian & Jungerwirth, 1990). However, the knowledge regarding the receptors involved for specific mosquito vector infection is minimal and often based on work in cell culture. Examinations of CHIKV in C6/36 cells suggest that infection of mosquito cells in vitro is mediated by a clathrin-dependent endocytic pathway (Lee et al., 2013). Using SINV, a possible receptor NRAMP (natural resistance-associated macrophage protein) was identified in both insect (Drosophila spp.) and mammalian hosts (Rose et al., 2011). Studies in C6/36, Drosophila melanogaster, and Anopheles stephensi have all identified heparin sulfate as a key player (Lin, Buff, Perrimon, & Michelson, 1999; Sakoonwatanyoo, Boonsanay, & Smith, 2006; Sinnis et al., 2007); however, other research has found that heparin sulfate does not play an essential role in DENV infection of mosquito cells (Hung et al., 2004; Thaisomboonsuk, Clayson, Pantuwatana, Vaughn, & Endy, 2005). Historically, enhanced utilization of heparin sulfate binding indicates viral adaptation to cell culture and heparin sulfate (Klimstra, Ryman, & Johnston, 1998; Kroschewski, Allison, Heinz, & Mandl, 2003), so this phenomenon may be biasing receptor studies. Virus overlay protein binding assays are emerging as a method for isolating membrane proteins, although the limitations of this method include lack of sensitivity as proteins of the same weight cannot be distinguished (Smith, 2012). This method has aided identification of a number of DENVs receptor candidates in C6/36 cells (Chu & Ng, 2004; Kuadkitkan, Wikan, Fongsaran, & Smith, 2010; Sakoonwatanyoo et al., 2006; Salas-Benito & del Angel, 1997; Salas-Benito et al., 2007; Yazi Mendoza, Salas-Benito, LanzMendoza, Hernandez-Martinez, & del Angel, 2002). Mendoza et al. recently characterized the binding of all four serotypes of DENV to Ae. aegypti organs including midgut, ovary, salivary gland, eggs, larvae,

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and pupae cell extract and identified a 45 kDa glycoprotein that has also been identified as expressed in C6/36 cells (Yazi Mendoza et al., 2002). A similar study investigating DENVs in Ae. aegypti and Aedes polynesiensis identified four distinct receptor candidates as compared to what had already been identified in cell culture for each mosquito species (Cao-Lormeau, 2009). Of these DENV studies the only protein that has been clearly identified as a receptor, prohibitin, has only been examined in mosquito cell lines (Kuadkitkan et al., 2010). Few other proposed insect cell receptors have been identified for Japanese encephalitis virus (JEV) (Boonsanay & Smith, 2007; Chu et al., 2005) and WNV (Chu et al., 2005; Xia & Zwiebel, 2006). Yet, recent findings proposing a new model of examining virus directly obtained from infected mosquitoes indicate that DENV and WNV may directly penetrate the host cell plasma membrane (Vancini, Kramer, Ribeiro, Hernandez, & Brown, 2013). This method of infection has also been proposed for alphaviruses as electron microscopy studies of immunolabeled SINV proteins at the BHK-21 cell plasma membrane showed an increase of empty viral particles with an elevation in temperature, but has yet to be examined in mosquito cells or vectors. The authors suggest this increase in temperature would curb endosome formation and membrane fusion and therefore entry of alphaviruses that likely occurs by direct penetration of the cell membrane (Vancini, Wang, Ferreira, Hernandez, & Brown, 2013).

3.3. Vector genetics that modulate viral infection Despite the potential for arboviruses to bypass a cellular receptor upon invasion, genetic differences between different mosquito vectors can result in drastic differences in vector susceptibility to infection with the same virus. For instance, the WR laboratory-selected Cx. tarsalis genetic variant was observed to be resistant to infection with WEEV, while another laboratory-derived genetic population (WS Cx. tarsalis) was selected to be extremely susceptible to oral infection with the same WEEV strain (see Hardy, Apperson, Asman, & Reeves, 1978; Hardy & Reeves, 1990). Similarly, WEEV has been determined to infect Cx. tarsalis mosquitoes efficiently in blood meals containing 3 log10 PFU/ml blood ingested, yet fails to infect Culex pipiens at blood meal titers in excess of 8 log10 PFU/ml (Hardy et al., 1976; Kramer, Hardy, Houk, & Presser, 1989). Furthermore, Tesh et al. have demonstrated minimal infection titers differing by as much


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as 1000-fold for geographic strains of Ae. albopictus infected with CHIKV (Tesh, Gubler, & Rosen, 1976). A number of studies have identified vector proteins and genes that likely add other variables to the vector competence. For instance, C-type lectins mosGCTL-1 and mosPTP-1 are critical for infection of Ae. aegypti and Cx. quinquefasciatus by WNV (Cheng et al., 2010). Utilizing quantitative inheritance methods, regions of Ae. aegypti that held determinants for susceptibility to DENV as well as variation in the midgut escape barrier were detected (Black et al., 2002; Bosio, Fulton, Salasek, Beaty, & Black, 2000). When these mosquito loci were quantified from different colonies and locations, it was discovered that their influence varied depending on geographic region and the style of laboratory management, which illustrates the complexity of the variables contributing to competence of a given vector. Nevertheless, advances in genome sequencing throughput and technology have facilitated the examination of individual mosquito genes in a truly quantitative way. To date, there have been many studies examining vector proteomics and gene expression as they pertain to infection (Behura et al., 2011; Bonizzoni et al., 2012; Chen, Mathur, & James, 2008; Colpitts et al., 2011; Girard et al., 2010; Tchankouo-Nguetcheu et al., 2012, 2010). One in which the authors analyzed transcriptome expression of Cx. quinquefasciatus, Ae. aegypti, and Anopheles gambiae in response to infection with WNV, Wuchereria bancrofti, and nonnative bacteria demonstrated the utility of such methods to generate unparalleled amounts of quantitative data for analysis. Through this study the authors were able to identify patterns of expression between the three vectors in response to pathogen infection and establish a set of genes to examine further (Bartholomay et al., 2010). In another application of this technology, Behura et al. compared genome-wide transcriptome profiles between susceptible and refractory populations of Ae. aegypti in response to DENV infection (Behura et al., 2011). Although the analysis of this data was complicated, they were able to identify differential expression in over 2000 genes, which punctuates the importance that mosquito population genetics likely plays in vector competence and the necessity to consider vector genetics as a variable in laboratory competence experiments.

3.4. Blood-feeding factors and vector infection Passage of Ross River virus and VEEV in mosquito cells has been demonstrated to result in the addition of glycosylation motifs on the surface of the

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E2 glycoprotein with high-mannose sugars that are poor inducers of type-I interferon secretion by myeloid dendritic cells compared to viruses grown in vertebrate cells possessing alternative carbohydrate modifications. This reduced induction of IFN-alpha/beta subsequently has been associated with increased viral replication of mosquito cell cultured viruses in vertebratederived myeloid dendritic cells, thus highlighting a potential mechanism for establishing infections in the intervening vertebrate hosts (Shabman et al., 2007). In addition to the differential glycosylation of the envelope proteins that dictate host immune response, the process of blood feeding can significantly affect the infection efficiency of arboviruses. The clotting process results in subsequent fluidic movement of the virus contained in the host sera to the periphery which places virions in close proximity to the apical surface of the midgut epithelia (Fig. 2.2, panel 1b) (Weaver, Lorenz, & Scott, 1993). A direct comparison of Ae. aegypti susceptibility to Mayaro virus infection by artificial membrane versus a viremic mouse indicated the natural blood meal method to be more infectious than the defibrinated infectious blood meal (Long et al., 2011); however, other studies have demonstrated a minimal effect of utilizing heparinized blood on mosquito infection rates (Mahmood, Chiles, Fang, & Reisen, 2004). Addition of polycations to infectious blood meals has been used to increase the per os midgut infection rate of Ae. aegypti mosquitoes with Semliki Forest Virus, SINV, and WNV, as well as for WEEV infection of Cx. pipiens (Houk et al., 1990). In contrast, neither YFV 17D strain in Ae. aegypti or SINV in An. stephensi demonstrated increased infection following the addition of dextran to the infectious blood meal (Pattyn & De Vleesschauwer, 1970). Further experiments have demonstrated midgut surface charge differences among mosquito species might play a role in midgut infection for some virus–vector interactions (Houk, Hardy, & Chiles, 1986). Although the mechanism dictating such differential effects of virus and mosquito midgut epithelial cells has not been elucidated, several studies have demonstrated that thawed virus utilized for mosquito infections result in lower infection rates than viruses grown directly in cell culture (Miller, 1987; Richards, Pesko, Alto, & Mores, 2007). It has been hypothesized that such differences could be related to altered receptor configuration on the surface of the freeze–thawed viruses. Additionally, a series of cascading mosquito physiological effectors have been documented to be modified by the act of blood feeding, including altered miRNA expression and subsequent gene expression profiles that could alter receptivity to arboviral infection and transmission (Hussain, Walker, et al., 2013).


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4. MIDGUT ESCAPE AND DISSEMINATION Failure of a virus to infect peripheral tissues after initiation of a productive infection of the midgut epithelium constitutes a midgut escape barrier. For an arthropod-borne virus to be transmitted, the virus must traverse the mesenteronal epithelium from the apical to basolateral side, exit the cell, and bypass the BL (Hardy, 1988; Hardy et al., 1983). The thickness of the BL has been postulated to be involved in the failure of flaviviruses and bunyaviruses to reach the mosquito hemocoel (Romoser et al., 2005; Thomas, Wu, Verleye, & Rai, 1993). Furthermore, it was demonstrated that nutritional status of larvae can modulate the thickness of that barrier (Grimstad & Walker, 1991; Jennings & Kay, 1999). It has been long established that architecture of the BL of the midgut epithelial cells would at most allow for a particle of 15 nm to permeate, so a much larger virion would theoretically be unable to escape through the BL (Reddy & Locke, 1990). However, Houk et al. demonstrated that viral particles are capable of circumvention of this size barrier (Houk, Hardy, & Chiles, 1981), although the mechanisms through which viruses bypass this potential barrier are not completely understood. Intrathoracic inoculation of mosquitoes with viruses having midgut escape barriers from per os challenge have developed midgut infections, indicating that the virus was capable of bypassing the BL from the basal side (Hardy et al., 1983; Nuckols et al., 2012; Romoser et al., 2004). Previously explored mechanisms of escape include passage through a “leaky midgut” (Hardy et al., 1983; Miles, Pillai, & Maguire, 1973; Weaver, 1986; Weaver et al., 1988; Weaver, Scott, Lorenz, & Repik, 1991), escape through the foregut/midgut junction (Mourya & Mishra, 2000; Romoser, Faran, & Bailey, 1987; Weaver et al., 1993, 1991), utilization of mosquito central nervous system (Miles et al., 1973), or dissemination through the established network of tracheae which penetrate the epithelial BL to deliver oxygen (Romoser et al., 2004; Volkman, 1997; Wigglesworth, 1977). While there has been no conclusive evidence demonstrating that viruses utilize these tracheoles as conduits for dissemination, there is considerable documentation that multiple arboviruses are able to infect tracheal branches (Bowers, Abell, & Brown, 1995; Chandler, Blair, & Beaty, 1998; Engelhard, Kam-Morgan, Washburn, & Volkman, 1994; Kirkpatrick, Washburn, Engelhard, & Volkman, 1994; Romoser et al., 2005, 2004; Smith, Adams, Kenney, Wang, & Weaver, 2008; Volkman, 1997). Recent work studying baculoviruses has strived to explain

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how virions can infect tracheoles as these cells are also lined with BL. Passarelli proposes that baculoviruses express a gene that activates a signal transduction pathway that stimulates BL lamina turnover, which could allow for the virus to escape (Passarelli, 2011). However, as most arboviruses have significantly fewer genes than baculoviruses, surmising that arboviruses might be also utilizing this mechanism would require more intense study.

4.1. Intrahost viral populations Arboviruses and their interactions with mosquito vectors display a complex interplay between RNA viruses and the innate immune response of mosquito vectors (Brackney, Beane, & Ebel, 2009). Arboviruses are almost exclusively comprised of RNA viral genomes. Having an error-prone RNA-dependent RNA polymerase and lacking proofreading function, these viruses have a remarkable capacity for generating viral variants that can adapt to changing replicative environments (Domingo, 1997). These variants form a heterogeneous population of related sequences that are referred to as quasispecies or a mutant swarm. Evolutionary selection seems to act on viral populations rather than the individual variants (Biebricher & Eigen, 2005; Eigen, 1993). Furthermore, in C6/36 cells, cooperative interactions of individual WNV variants may enhance swarm fitness levels so that they surpass the relative fitness levels of any individual genotype in vitro (Ciota, Ehrbar, Van Slyke, Willsey, & Kramer, 2012). It has also been determined that viruses with artificially increased replication fidelity yield a less diverse viral population and are also likely to be attenuated (Vignuzzi, Wendt, & Andino, 2008). This has been shown specifically with a CHIKV strain with a high-fidelity polymerase that generated a viral population with a reduced genetic diversity and had a deleterious effect in both the invertebrate and vertebrate host (Coffey, Beeharry, Borderia, Blanc, & Vignuzzi, 2011). Studies designed to understand why SLEV activity and geographic range has been relatively restrained in comparison to WNV examined the size, composition, and phylogeny of intrahost swarms for each virus strain from mosquito isolates and discovered a general trend of a loss of intrahost diversity in time and by location in SLEV. The authors suggest that this loss of host viral population diversity has constrained SLEV activity in comparison to WNV (Ciota, Koch, et al., 2011). Higher viral genetic diversity has been identified in WNV populations grown strictly in mosquito cells, while genetic diversity has been observed to be restricted in viruses propagated in avian vertebrate cells ( Jerzak, Brown, Shi, Kramer, & Ebel, 2008).


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Presumably, these differences in viral diversification are the result of the differential effects of adaptive responses for combating the effects of antiviral (RNAi) response in mosquitoes (Brackney et al., 2009) and the strong purifying selective effects imposed by the avian host (Deardorff et al., 2011). The theory that a homogenous intrahost viral population may decrease viral fitness levels has particular bearing on dual-host viruses like arboviruses. Despite the aforementioned inherent capacity for mutation and subsequent adaptation, arboviruses have demonstrated nucleotide substitution rates of approximately 104 nucleotide substitutions per base pair per year (Weaver et al., 1994), which is lower than that observed in viruses that utilize single hosts ( Jenkins, Rambaut, Pybus, & Holmes, 2002). These differing evolutionary rates coupled with empirical data from viral passaging studies have led to the theory that the alternating cycles between mosquito and vertebrate hosts results in a compromised fitness level for the virus in either of the hosts. Numerous in vitro (Greene et al., 2005; Moutailler et al., 2011; Vasilakis et al., 2009; Weaver, Brault, Kang, & Holland, 1999) and in vivo (Ciota, Ehrbar, Matacchiero, Van Slyke, & Kramer, 2013; Ciota et al., 2009; Ciota, Styer, Meola, & Kramer, 2011; Coffey et al., 2008; Deardorff et al., 2011) studies have been performed with various arboviruses in which the requirement to replicate in both hosts has been eliminated in order to assess the potential that replication in the two disparate hosts restricted viral diversification. Data from the various studies have supported (Coffey et al., 2008) as well as contradicted (Ciota et al., 2009, 2008; Deardorff et al., 2011) this hypothesis. The preponderance of data suggests that arboviruses are exposed to strong purifying selective pressures and that viral diversification and fitness changes can be disproportionately induced, but are dependent upon the host system assessed. For instance, when WNV and SLEV were released from their dual-host cycle and serial passaged through intrathoracic inoculation of Cx. pipiens, WNV acquired enhanced replication in mosquitoes without an apparent replication cost in chickens. However, serial intrathoracic passage of SLEV through Cx. pipiens yielded a mildly attenuated growth phenotype in mosquitoes (Ciota et al., 2008). Recognizing the potential bias for WNV to SLEV based on the preestablished limited intrahost population diversity for SLEV, these authors evaluated the capacity of SLEV to acquire adaptive mutations when released from the dual cycle and identified a lack of consensus sequence change following serial passage in mosquitoes or chickens. The authors concluded that constraints on arbovirus fitness in nature were not

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solely due to limitations of persisting in a dual-host cycle (Ciota et al., 2009). Fitness of these populations appeared to be largely due to the size diversity of the intrahost viral population, which is driven by an error-prone polymerase.

4.2. Viral population bottlenecks In contrast to intrahost genetic diversity arising in mosquitoes resulting from section pressures afforded by exposure to a strong RNAi response (Brackney et al., 2009), population bottlenecks at the midgut entry and escape, and the salivary entry and escape barriers (Coffey et al., 2008) have been investigated as a source of genetic constraint. A genetic bottleneck consists of a marked decrease in the population size and can result in a loss of fitness by way of Muller’s Ratchet due to the accumulation of largely deleterious mutations (Duate, Clarke, Moya, Domingo, & Holland, 1992; Muller, 1964). Bottlenecks have been described between insect transmission of plant viruses suggesting that transmission bottlenecks may be important for mosquitoborne viruses as well (Ali et al., 2006; Moury, Fabre, & Senoussi, 2007). A bottleneck upon midgut infection where only a few cells are initially infected has been described for epizootic VEEV in Ae. taeniorhynchus (Smith et al., 2008), WNV in Cx. quinquefasciatus (Scholle, Girard, Zhao, Higgs, & Mason, 2004) and Cx. pipiens (Ciota, Ehrbar, Van Slyke, Payne, et al., 2012) as well as for SINV in Ae. aegypti (Myles, Pierro, & Olson, 2004). Interestingly, studies looking at enzootic VEEV in their vector, Culex taeniopus, indicate that with low doses, there is a bottleneck upon midgut infection; however, when the infectious dose is higher, the more severe bottleneck occurred at the midgut escape (Forrester, Guerbois, Seymour, Spratt, & Weaver, 2012; Kenney et al., 2012). This suggests that for enzootic VEEV and potentially other viruses as well, the point and severity of the initial bottleneck may be dependent upon exposure dose. Potential salivary gland infection barriers for some vectors of WNV, RVFV, JEV, VEEV, and WEEV have also been implicated by a number of experimental vector competence studies (Lutomiah et al., 2011; Mahmood, Chiles, Fang, Green, & Reisen, 2006; Turell, Dohm, Fernandez, Calampa, & O’Guinn, 2006; Turell et al., 2007; Turell, Mores, et al., 2006; Turell, Wilson, & Bennett, 2010). Much like previously described midgut infection barriers, salivary gland barriers for a given virus appear to differ to varying degrees based on the specific vector species and population being examined.


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4.3. Physiological and pathological changes imparted by arboviral infection It has long been assumed that the ideal symbiosis between an arbovirus and its insect vector likely evolves toward a mutualistic relationship; however, there are many instances in which one can alter the biology of the other. Arboviral infection of mosquito vectors has been associated with altered mosquito behavior. For instance, orally or vertically infected female Aedes triseriatus mosquitoes have demonstrated higher insemination rates than uninfected age-matched females when mixed with males (Gabitzsch, Blair, & Beaty, 2006; Reese, Beaty, Gabitzsch, Blair, & Beaty, 2009). Furthermore, infection of Ae. aegypti mosquitoes with DENV has been associated with longer probing and feeding times that could increase the likelihood of viral transmission. The exact mechanism(s) for these increased probing and feeding times have not been determined; however, DENV infection of accessory organs associated with blood feeding were positively correlated with the phenotype (Platt et al., 1997). Pathological changes in the midgut of the enzootic vector of eastern equine encephalitis virus (EEEV), Culiseta melanura, have been associated with increased dissemination efficiency (Weaver & Scott, 1990; Weaver et al., 1988). Pathological changes in the midgut epithelia of highly susceptible Cx. tarsalis mosquito colonies have been observed to be more prominent following WEEV infection than those observed in more refractory mosquito populations, thus indicating that vector competence of mosquitoes could also be associated naturally with differential viral-induced pathological responses (Weaver, Lorenz, & Scott, 1992). Although alphavirus infection is more likely to be associated with vector pathology (Lambrechts & Scott, 2009), studies of WNV in Culex spp. mosquitoes have demonstrated apoptotic changes in the midgut and salivary glands (Girard, Popov, Wen, Han, & Higgs, 2005; Girard et al., 2007; Vaidyanathan & Scott, 2006). In theory, deleterious effects of arboviruses to vector fitness are more likely to occur when the virus and vector are not well adapted to each other. However, a WNV strain that had been serially passaged 20 times and considered “mosquito adapted” resulted in decreased Cx. pipiens survival and altered fecundity (Ciota et al., 2013), which would suggest that “adaptation” does not necessarily lead to a commensal relationship, or that more than 20 passages are required for coevolution to occur. An earlier study demonstrating that EEEV reduces survival and reproduction of Cs. melanura, also showed that viruses recovered greater than 50 years later did not show any measurable attenuated effect in the vector indicating that any coevolution occurring in 50 years

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of EEEV cycling did not result in a more symbiotic relationship between the virus and vector (Scott & Lorenz, 1998). Similar studies examining fitness changes in Cx. tarsalis females following infection with WEEV as compared to uninfected controls demonstrated a reduction in fitness (Mahmood, Reisen, Chiles, & Fang, 2004). Interestingly, recent examinations of the potential fitness costs of WNV infection in a highly susceptible Cx. pipiens colony showed that WNV infection did not alter mosquito fecundity or blood feeding. However, findings did indicate that resistance to infection is associated with a fitness cost in mosquito survival (Ciota, Styer, et al., 2011). A meta-analysis suggested that overall arboviruses do reduce the survival of their mosquito vectors; however, the extent to which this occurs is highly dependent on the vector–virus taxonomy and interaction. For instance, horizontally maintained virus cycles were correlated with increased likelihood of virus-induced mortality, whereas transovarial maintained bunyaviruses were unlikely to cause deleterious effect in Aedes spp. mosquitoes (Lambrechts & Scott, 2009).

5. ENVIRONMENTAL VARIABLES When considering the competence of a vector to transmit a given virus, there are several environmental factors that can have drastic effects. The most abundantly studied is temperature as it can alter vector competence in a number of ways. Many studies indicate that temperature along with competition during the larval stage of development may be correlated with a competence phenotype (Alto, Lounibos, Mores, & Reiskind, 2008; Kay, Fanning, & Mottram, 1989; Kramer et al., 1983; Mourya, Yadav, & Mishra, 2004; Muturi & Alto, 2011). Similarly, temperature can have a significant impact on vectorial competence of an adult mosquito. Changes in temperature during the EIP have been repeatedly shown to affect the efficiency of viral dissemination and transmission (Kramer et al., 1983; Lambrechts et al., 2011). Typically, it has been accepted that as the ambient temperature increases, virus replication will increase in mosquito tissues (Reisen et al., 2006; Reisen, Meyer, Presser, & Hardy, 1993). Recent trends in seasonal WNV in the United States seem to support the contribution of temperature as well as drought. Scrutiny of temperature and precipitation variations on seasonal mosquito abundance and the prevalence of WNV in the northeastern United States found a positive association between drought and increases in mosquito infection rates in 2010 when compared with 2011, which was milder and wetter. The authors suggested that there


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are clear temperature and precipitation thresholds that predict subsequent outbreak levels of WNV ( Johnson & Sukhdeo, 2013). Furthermore, studies have demonstrated that wide fluctuations of diurnal temperature range in the poikilothermic mosquito vector can facilitate dissemination of DENV in Ae. aegypti (Carrington, Armijos, Lambrechts, & Scott, 2013). Studies examining climate change patterns predict that there will be increases in weather extremes such as larger precipitation events as well as longer intervals between these events (Groisman & Easterling, 1994; Karl, Knight, Easterling, & Quayle, 1996; Knapp et al., 2008). Such changes are likely to affect the abundance of breeding habitats of various arbovirus vectors based on the oviposition preferences of the vector species.

6. MOSQUITO-SPECIFIC VIRUSES Cell-fusing agent virus was first isolated in Ae. aegypti cell culture and identified by genomic sequence analyses to be similar in genome organization and identity to other members of the family Flaviviridae (CammisaParks, Cisar, Kane, & Stollar, 1992). Similar viruses (termed “insect-specific flaviviruses”; ISFs) were not known to circulate in nature until the identification of Kamiti River virus from field-collected Aedes macintoshi mosquitoes in Kenya in 2003 (Sang et al., 2003). In subsequent studies, other apparent mosquito-borne flaviviruses have been identified (Culex flavivirus; CxFV) in Cx. pipiens mosquitoes in Japan (Hoshino et al., 2007) and Aedes spp. mosquitoes (Aedes flavivirus) in Puerto Rico (Cook et al., 2006). A mosquitospecific flavivirus has recently been identified in Cx. tarsalis mosquito pools in California (Kern County) as well as western Canada and Colorado (Tyler et al., 2011). Little is known regarding the mechanisms by which ISFs are transmitted; however, the fact that these viruses fail to replicate or elicit cytopathic effects in mammalian cells (Blitvich et al., 2009; Bolling, Eisen, Moore, & Blair, 2011; Bolling, Olea-Popelka, Eisen, Moore, & Blair, 2012; Sang et al., 2003; Tyler et al., 2011) indicates the likelihood that these agents are transmitted solely among mosquitoes. As such, it is highly likely that mosquitoes are infected transovarially (virus within egg) or transovularilly (virus on egg). Field evidence of ISF-infected immature mosquitoes (Bolling et al., 2012; Saiyasombat, Bolling, Brault, Bartholomay, & Blitvich, 2011) and males (Bolling et al., 2011) further support this assertion. In one study, larvae reared from Cx pipiens isofemale lines demonstrated 100% vertical infection rate with 97.4% filial infection rate with CxFV (Saiyasombat et al., 2011). Mosquitoes infected with ISFs would therefore

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likely be infected from emergence and could be positive for this virus prior to exposure to alternative flaviviruses, such as WNV (Newman et al., 2011). Identifying the mechanism(s) of transmission and its tissue distribution within infected Culex spp. mosquitoes will be imperative in order to assess the potential for this virus to inhibit WNV infection and/or transmission (see below). Additionally, this viral agent could be useful for expression of heterologous genes designed at inhibiting replication of alternative flaviviruses. For instance, transient transduction of Ae. aegypti using a modified SINV system has effectively been used to express double-stranded DENV RNA resulting in the failure of these mosquitoes to transmit this alternative virus (Olson et al., 1996).

6.1. Superinfection exclusion Barriers to superinfection have been described previously for arthropodborne viruses (Beaty, Sundin, Chandler, & Bishop, 1985; Davies, Jones, & Nuttall, 1989; Eaton, 1979; el Hussein, Ramig, Holbrook, & Beaty, 1989; Karpf, Lenches, Strauss, Strauss, & Brown, 1997; Singh, Suomalainen, Varadarajan, Garoff, & Helenius, 1997; Sundin & Beaty, 1988). For example, Ae. triseriatus mosquitoes experimentally infected with LACV have been shown to be resistant to infection with another closely related bunyavirus, Snowshoe Hare virus, approximately 2 days postinfection (Beaty et al., 1985). A resistance period for dual infection was identified for ticks coinfected with Thogoto virus (THOV) for a period ranging between 24 h and 10 days of the initial infection with an alternative THOV strain (Davies et al., 1989). An Ae. albopictus cell (C6/36) model further demonstrated that the inhibition of the secondarily infecting alphavirus is sequence specific. Different alphaviruses were blocked from superinfection of C6/36 cells, while unrelated bunyaviruses or flaviviruses could establish infection in alphavirus-infected C6/36 cells within the time period that these cells were resistant to infection with heterologous alphaviruses (Eaton, 1979; Karpf et al., 1997). The mechanism for this “superinfection exclusion” has not been identified; however, it has been hypothesized that competitive exclusion through template scavenging during RNA replication as well-incompatible interactions between viral proteins exclude replication of the secondarily infecting virus. Inhibitory effects on the superinfecting virus have been identified in the processes of binding to the cellular surface receptors, low-pH fusion with the endocytic vesicle, viral uncoating, viral replication as well as viral maturation and budding (Singh


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et al., 1997). Given the relatedness of the recently described ISFs isolated from Californian Cx. tarsalis mosquitoes to WNV, a potential similar barrier to superinfection with heterologous flaviviruses could reduce susceptibility of ISF-infected mosquitoes to WNV infection and/or for transmission. Such information would be critical for predicting areas of importance for WNV transmission for mosquito abatement efforts as well as for the development of mechanisms to block WNV transmission through biological control strategies. Several studies have directly assessed the potential for CxFV to directly block infection and transmission capacity of Culex spp. mosquitoes with WNV (Bolling et al., 2012; Kent, Crabtree, & Miller, 2010). When Cx. quinquefasciatus mosquitoes were intrathoracically inoculated with CxFV and orally exposed to WNV, no reduction in oral infectivity or transmissibility was observed. In contrast, when a CxFV persistently infected colony of Cx. pipiens was assessed for oral infectivity with WNV, Bolling et al. observed a moderate suppression of early replication in exposed mosquitoes (Bolling et al., 2012). A study with a newly described ISF designated Palm Creek virus has demonstrated a capacity for reducing viral replication of both Kunjin and Murray Valley encephalitis viruses in coinfected C6/36 cells (Hobson-Peters et al., 2013). Despite the potential for superinfection exclusion of medically important viruses by ISFs, a positive correlation between WNV and CxFV infection of Cx. pipiens mosquitoes in Illinois has been observed, suggesting that there could be a biological interaction between these viruses such as RNAi suppression that could mediate increased susceptibility in naturally infected mosquitoes to WNV (Newman et al., 2011).

6.2. Vertical transmission of arboviruses in mosquitoes Transmission of ISFs is limited to vertical transmission, whereas the primary method of transmission for arboviruses of public health importance is via the oral route. However, a number of dual-host arboviruses have been found to utilize a secondary vertical maintenance cycle in addition to their wellestablished dual-host cycle (Aitken, Tesh, Beaty, & Rosen, 1979; Rosen, Lien, Shroyer, Baker, & Lu, 1989; Rosen, Shroyer, Tesh, Freier, & Lien, 1983; Tesh, 1984). Some viruses with poorly understood human health impacts have a well-established record of transovarial transmission. Specifically, many bunyaviruses in the California encephalitis serogroup such as LACV and California encephalitis virus (Miller, DeFoliart, & Yuill, 1977; Tesh & Gubler, 1975; Turell, Hardy, & Reeves, 1982; Turell, Reeves, &

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Hardy, 1982), Jamestown Canyon virus (Hardy, Eldridge, Reeves, Schutz, & Presser, 1993), and many others (Dutary, Petersen, Peralta, & Tesh, 1989; Reisen et al., 1990) have been found to utilize a vertical transmission cycle in multiple vectors. It is largely believed that these cycles allow the virus to perpetuate a minimum infection rate in a mosquito population throughout periods of low or no natural dual-host cycling. Vertical transmission of DENV 1–4 (Aitken et al., 1979; Joshi, Mourya, & Sharma, 2002; Mourya et al., 2001) and YFV (Aitken et al., 1979; Beaty, Tesh, & Aitken, 1980) have been repeatedly shown in the lab and the field (Angel, Sharma, & Joshi, 2008; Le Goff et al., 2011; Martins et al., 2012; Vilela et al., 2010), despite the highly successful anthropozoonotic dual-host cycle of these viruses. A recent study examining field-collected larvae in the dry season and wet season suggested that more vertical transmission is occurring during the wet season and likely important for maintenance of DENV 1 and 2 infection throughout the dry season in Indonesia (Mulyatno, Yamanaka, Yotopranoto, & Konishi, 2012). Other studies have examined the importance of vertical transmission in WNV indicating that Cx. tarsalis, Cx. pipiens, and Culex salinarius can transmit virus transovarially to their offspring (Anderson, Main, Cheng, Ferrandino, & Fikrig, 2012; FechterLeggett, Nelms, Barker, & Reisen, 2012). Studies of the contribution of vertical transmission to overwintering of WNV in Culex spp. in California demonstrated that while Cx. tarsalis showed a field vertical transmission rate of 26%, transstadial transmission was lost in Cx. pipiens 75% of the time indicating that while vertical transmission of WNV is common in California, maintenance may not be that efficient (Nelms et al., 2013). A virus with more sporadic emergence, SLEV, has long been demonstrated to utilize transovarial transmission (Chamberlain, Sudia, & Gogel, 1964; Flores, Diaz, Batallan, Almiron, & Contigiani, 2010; Francy, Rush, Montoya, Inglish, & Bolin, 1981; Hardy, Rosen, Reeves, Scrivani, & Presser, 1984; Nayar, Rosen, & Knight, 1986; Pelz & Freier, 1990; Reisen et al., 2002); however, whether vertical transmission is required for SLEV to persist between periods of epizootics as well as overwintering is less clear.

7. UTILIZING MOSQUITO BIOLOGY TO INHIBIT ARBOVIRUS INFECTION 7.1. Mosquito innate immune response RNAi pathways in mosquitoes encompass at least three systems including miRNA, small-interfering (siRNA), and Piwi-interacting (Hess et al.,


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2011) gene-silencing pathways (Blair, 2011). In addition to targeting double-stranded RNA indicative of viral infections, these pathways are involved in a diverse range of functions from altering gene expression during ontological development to reducing transposable element chromosomal integration. Functionally, this pathway serves as the primary immune response of mosquitoes against arboviruses through the activation of pathogen-associated molecular patterns. With arboviral infections, these recognition elements encompass double-stranded RNA that serves as a replicative intermediate for RNA viruses. A thorough review of the RNAi response in mosquitoes is provided in Blair (2011). Studies investigating the genetic diversity of these silencing pathways in exemplar arboviral vectors such as Cx. pipiens and Ae. aegypti as well as nontypical arboviral vectors such as Anopheles gambiae have demonstrated a higher rate of evolution of the gene components involved in these silencing pathways for key arboviral vectors as opposed to nonarboviral vector mosquitoes (Campbell, Black, Hess, & Foy, 2008). In addition, intraspecific comparisons of these gene components in Ae. aegypti have demonstrated that siRNA gene-silencing pathways evolve at a disproportionate rate and evolve under the effects of positive selection compared to all other genes, thus providing indirect evidence that mosquito infection of RNA viruses can serve as a strong selective pressure with diversification of mosquito hosts (Bernhardt et al., 2012). Reciprocally, experimental evidence of WNV subjected to sequential mosquito passages has demonstrated that portions of the viral genome most commonly targeted by the RNAi pathway are associated with high genetic polymorphisms (Brackney et al., 2009). The utility of the RNAi response in mosquito cells has further been demonstrated by the finding that Ae. albopictus C6/36 cells lack a functional RNAi pathway, thus contributing to the high susceptibility of this cell line for arboviral production (Brackney et al., 2010). Interestingly, climatic factors such as cooler temperatures have been observed to reduce the RNAi activity in mosquitoes (Adelman et al., 2013), thus allowing for higher infection rates with arboviruses. Thermal differences, therefore, can have the effect at one extreme of reducing the duration of the EIP at high temperatures while reducing the ability of a mosquito’s innate immune system to thwart an arboviral infection at lower temperatures. In addition to serving as a component of the innate immune response of mosquitoes to arboviral infections, a potential role of delivery and targeting critical small RNAs through siRNA silencing mechanisms could be employed as a new method for disrupting key metabolic functions in mosquitoes and a mosquitocidal treatment (Lucas, Myles, & Raikhel,

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2013). The RNAi pathway affords a target for arboviruses to antagonize in order to circumvent the predominant antiviral response in mosquitoes. This is performed through the expression of a series of virus-encoded RNAsilencing suppressor (RSS) proteins. The recently described subgenomic flavivirus RNA (sfRNA) generated by the incomplete 50 –30 exonuclease degradation of the flavivirus genomic transcript through to the 30 UTR (Funk et al., 2010) has demonstrated the capacity to alter XRN1 activity and mosquito mRNA profiles (Moon et al., 2012). Subsequent studies have demonstrated the capacity of this flaviviral sfRNA to disrupt both miRNA and siRNA-induced RNAi pathways, inhibiting double-stranded RNA by dicer (Schnettler et al., 2012), thus serving as an example of an arboviral RSS targeting the RNAi response in mosquitoes. WNV encodes a miRNA-like small RNA in the 30 -untranslated region that has been associated with an upregulation of GATA4 mRNA and subsequent facilitation of WNV replication in mosquito cells (Hussain et al., 2012). Wolbachia infection of Ae. aegypti has also been associated with the specific expression of aae-miR-12, an miRNA associated with the negative regulation of DNA replication (MCM6), and monocarboxylate transporter (MCT1) (Osei-Amo, Hussain, O’Neill, & Asgari, 2012). While RNAi-mediated antiviral effects appear to serve the dominant role as mediators of the antiviral innate immune response in mosquitoes, other innate immune pathways such as the Toll–Imd, Jak–STAT, Nf-kB, and autophagy pathways have demonstrated important roles in antiviral defense. It is becoming increasingly more apparent that the innate immune pathways of mosquitoes are interlinked and could result in targeted, pathogen-specific, and systemic responses to arboviral infections (Kingsolver, Huang, & Hardy, 2013). Such interactions have proven to be critical for antiviral effects against WNV such as the interaction between the Jak–STAT and Nf-kB.

7.2. Microbiota In contrast to parasitic infections of mosquito vectors (Weiss & Aksoy, 2011), relatively few studies have assessed the effect of midgut microbiota on arboviral competence. One study, however, demonstrated that Ae. aegypti cleansed of gut flora by antibiotic treatment had increased susceptibility to DENV-2 infection (Xi et al., 2008). Although the mechanisms of this increased susceptibility have not been elucidated, Toll pathway innate immune responses stimulated by the presence of gut microbiota have been


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implicated in the differences in viral loads (Xi et al., 2008). Studies using Drosophila genetic mutants lacking functional Toll and Imd pathways have failed to implicate this pathway with Wolbachia-induced refractoriness of Drosophila to DENV infection (Rances et al., 2013). Furthermore, specific observations have subsequently been made that endosymbiont Serratia odorifera enhances susceptibility of Ae. aegypti to DENV-2 (ApteDeshpande, Paingankar, Gokhale, & Deobagkar, 2012). Moreover, a study performed with LACV incubated with bacterial cultures isolated from Ae. albopictus demonstrated reduced in vitro infectivity. This finding implied that extracellular factor(s) released by the microbiota could impede viral infectivity of the cells ( Joyce, Nogueira, Bales, Pittman, & Anderson, 2011) as the bacteria were removed prior to exposure of cells. Introduction of nonnative microbiota such as the Gram-negative bacterium Wolbachia has induced reduced susceptibility of Ae. aegypti to DENV (Blagrove, Arias-Goeta, Failloux, & Sinkins, 2011), WNV (Hussain, Lu, et al., 2013), and CHIKV (Blagrove, Arias-Goeta, Di Genua, Failloux, & Sinkins, 2013). Similar studies performed with Ae. albopictus demonstrated no effect on transmissibility of CHIKV but significantly reduced transmission rates of this mosquito for DENV (Mousson et al., 2012). Wolbachia endosymbionts of Cx. quinquefasciatus have also been associated with decreased susceptibility to WNV (Glaser & Meola, 2010). Interestingly, studies with Wolbachia in Ae. aegypti cells (Aag2) demonstrated higher levels of accumulated viral RNA than in Aag2 cells not exposed to Wolbachia; nevertheless, the levels of secreted virus were significantly lower. In vivo infection of Wolbachiapositive Ae. aegypti demonstrated strain specificity of the Wolbachia response as well as reduced transmissibility of mosquitoes intrathoracically or orally exposed to WNV (Hussain, Lu, et al., 2013). The high transmissibility of Wolbachia endosymbionts could lead to the development of a novel avenue for imparting resistance in mosquito populations to a series of arboviruses (Walker et al., 2011). Studies in Ae. aegypti infected with Wolbachia have demonstrated the direct effects of miRNA-induced gene regulation for pathways that could modulate viral replication (Osei-Amo et al., 2012). Further studies are warranted to assess this research avenue further in order to determine if this lies at the heart of the specificity of the inhibitory response to different arboviruses with Wolbachia strains. A number of questions have arisen from the aforementioned studies: namely, what are the inherent mechanism(s) that dictate the reduced vector competence of mosquitoes infected with Wolbachia strains? Insight into this mechanism will undoubtedly shed light on the basis of the differential effectiveness of Wolbachia

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strains as well on the grounds of the heterotypic effectiveness against different arboviral agents. The fact that mosquito culture systems demonstrate no retardation of viral transcription strongly indicates a posttranscriptional regulation to be a likely candidature for this inhibitory phenomenon. Nevertheless, the underlying mechanism(s) for such inhibition serve as an additional example to underscore the inherently complex interactions that exist between virus, vector, and the environment in which they coexist.

8. CONCLUSIONS The sections of this chapter have been designed to highlight and illustrate examples of the inherent complexity of the interactions between virus, vector, and the environment. Far from being “flying syringes,” there is a complex interplay between the innate immune system of mosquitoes and viral replication strategies (Schnettler et al., 2013, 2012). These interactions are further compounded through environmental factors such as the presence of different gut microbiota populations, alternative temperature, and the presence of alternative viruses to name but a few factors that provide unique selective pressures for arboviruses infecting mosquitoes. Furthermore, different arboviruses continually evolve at the population level due to exposure to these various selective pressures that are further complicated by transmission strategies for single or multiple mosquito vectors. Environmental factors such as the presence of genetically related viruses and microbiota can have generalized or specific effects on certain mosquito–viral interactions that dramatically alter vector competence needed for maintaining horizontally transmitted arboviruses in the field. Manipulation of facets of these complex interactions should provide direction for experimental studies for interfering with arboviral transmission in mosquitoes.

ACKNOWLEDGMENTS We would like to thank Dr. William K. Reisen for reading of the chapter and the incorporation of numerous helpful comments and Drs. Scott Weaver and Rebekah Kading for helpful suggestions regarding the mosquito figure generated.

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