Kenney and Brault 2014

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: abrault@cdc.gov
Contents
1. Background 40
2. Vectorial Capacity 42
3. Oral Vector Infection 44
3.1 Viral determinants of infection 47
3.2 Receptor-mediated midgut infection 49
3.3 Vector genetics that modulate viral infection 50
3.4 Blood-feeding factors and vector infection 51
4. Midgut Escape and Dissemination 53
4.1 Intrahost viral populations 54
4.2 Viral population bottlenecks 56
4.3 Physiological and pathological changes imparted by arboviral infection 57
5. Environmental Variables 58
6. Mosquito-Specific Viruses 59
6.1 Superinfection exclusion 60
6.2 Vertical transmission of arboviruses in mosquitoes 61
7. Utilizing Mosquito Biology to Inhibit Arbovirus Infection 62
7.1 Mosquito innate immune response 62
7.2 Microbiota 64
8. Conclusions 66
Acknowledgments 66
References 66
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 # 2014 Elsevier Inc. 39

ISSN 0065-3527 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800172-1.00002-1
40 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 genet-
ics, 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 meth-
odologies 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 bio-
chemical 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 transmit-
ted 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 pri-
mate. 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 fam-
ilies 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 infec-
tion of additional vectors to propagate the viral cycle. In addition to dual-
host (vertebrate and vector-infecting viruses) mosquito-borne viruses, a
number of mosquito-specific viruses have been identified for which the
Arboviral Interactions with Mosquitoes 41
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 ver-
tebrate 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 pass-
ing 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 transmit-
ted 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, dis-
semination 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
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 vec-
tors 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 pro-
ductive 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 envi-
ronmental (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 phys-
iological, ecological, and environmental factors relating vector, host, and
Figure 2.1 Diagram depicting the interaction between the vector, virus, and environ-
mental 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 trans-
missibility 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 mos-
quito, virus, and environmental variables in Fig. 2.1; brown centroid compos-
ite of all three variables. The concept of vectoral capacity includes factors other
than those associated with direct biological interplay between virus and mos-
quito. Myriad factors can strongly influence vectorial capacity such as mos-
quito 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
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 mos-
quito density (Miller, Monath, Tabachnick, & Ezike, 1989) and/or elevated
host titers (Komar, 2003; Turell et al., 2005). Seasonal variations in the sus-
ceptibility 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 compe-
tency 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, dif-
ferences 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 sub-
sequent 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 physiolog-
ical 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 under-
taken. 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
Figure 2.2 Diagram of mosquito anatomy designating the different barriers to infec-
tion, 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 dem-
onstrating 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 receptor-
mediated fashion at the apical surface and subsequently replicate within
the cell. A number of physiological changes also occur in the gut follow-
ing blood feeding that can alter the receptivity of the mosquito midgut
to viral infection such as exposure of the virions to trypsin and chymotryp-
sin, 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
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 follow-
ing 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 post-
transcriptional 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). Fur-
thermore, 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 receptive-
ness for replication. For instance, DENV-2 infection of Ae. aegypti dem-
onstrated modulations in up to 32 different miRNAs, many of which
have been implicated with host transcriptional regulatory and signal trans-
duction patterns known to be involved in viral replication and dissemina-
tion (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 temper-
ature 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 mem-
brane 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 recep-
tors on the apical surface of the epithelia of infection susceptible mosquitoes
(Hardy et al., 1983).
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 differ-
ent 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 deter-
mined 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 post-
blood 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 num-
ber and easily identified. For instance, following the La Réunion island out-
break 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
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 trans-
mitted 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 experi-
ments 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 struc-
tural 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 bun-
yaviruses 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,
2007), Crabtree et al. were able to demonstrate that deletions of the individ-
ual 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 spe-
cific 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 path-
way (Lee et al., 2013). Using SINV, a possible receptor NRAMP (natural
resistance-associated macrophage protein) was identified in both insect (Dro-
sophila spp.) and mammalian hosts (Rose et al., 2011). Studies in C6/36, Dro-
sophila 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 hep-
arin sulfate (Klimstra, Ryman, & Johnston, 1998; Kroschewski, Allison,
Heinz, & Mandl, 2003), so this phenomenon may be biasing receptor stud-
ies. Virus overlay protein binding assays are emerging as a method for iso-
lating 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, Lanz-
Mendoza, 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,
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 iden-
tified 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 immuno-
labeled 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 pen-
etration 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 inva-
sion, 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). Sim-
ilarly, WEEV has been determined to infect Cx. tarsalis mosquitoes effi-
ciently 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
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 sus-
ceptibility 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 loca-
tions, it was discovered that their influence varied depending on geographic
region and the style of laboratory management, which illustrates the com-
plexity of the variables contributing to competence of a given vector.
Nevertheless, advances in genome sequencing throughput and technol-
ogy 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 appli-
cation of this technology, Behura et al. compared genome-wide trans-
criptome 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 differ-
ential 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 demon-
strated to result in the addition of glycosylation motifs on the surface of the
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 vertebrate-
derived 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 pro-
teins that dictate host immune response, the process of blood feeding can
significantly affect the infection efficiency of arboviruses. The clotting pro-
cess 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 dem-
onstrated a minimal effect of utilizing heparinized blood on mosquito infec-
tion 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 differ-
ences 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 mid-
gut 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 physio-
logical effectors have been documented to be modified by the act of blood
feeding, including altered miRNA expression and subsequent gene expres-
sion profiles that could alter receptivity to arboviral infection and transmis-
sion (Hussain, Walker, et al., 2013).
4. MIDGUT ESCAPE AND DISSEMINATION

Failure of a virus to infect peripheral tissues after initiation of a produc-
tive 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 archi-
tecture of the BL of the midgut epithelial cells would at most allow for a par-
ticle 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 under-
stood. Intrathoracic inoculation of mosquitoes with viruses having midgut
escape barriers from per os challenge have developed midgut infections, indi-
cating 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 dis-
semination 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 dissemina-
tion, 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
how virions can infect tracheoles as these cells are also lined with BL. Pas-
sarelli proposes that baculoviruses express a gene that activates a signal trans-
duction pathway that stimulates BL lamina turnover, which could allow for
the virus to escape (Passarelli, 2011). However, as most arboviruses have sig-
nificantly 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 mos-
quito 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 inter-
actions 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 deter-
mined 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 inverte-
brate 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 compar-
ison 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).
Presumably, these differences in viral diversification are the result of the

differential effects of adaptive responses for combating the effects of ant-
iviral (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 uti-
lize single hosts ( Jenkins, Rambaut, Pybus, & Holmes, 2002). These differ-
ing 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
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). Bottle-
necks have been described between insect transmission of plant viruses
suggesting that transmission bottlenecks may be important for mosquito-
borne 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 vec-
tor, 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 sever-
ity 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.
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 prob-
ing 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 associ-
ated 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 asso-
ciated 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 refrac-
tory mosquito populations, thus indicating that vector competence of mos-
quitoes 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. How-
ever, 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 atten-
uated effect in the vector indicating that any coevolution occurring in 50 years
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 col-
ony 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, hor-
izontally 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 compe-
tence 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 sig-
nificant impact on vectorial competence of an adult mosquito. Changes in
temperature during the EIP have been repeatedly shown to affect the effi-
ciency 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
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 exam-
ining 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 organiza-
tion and identity to other members of the family Flaviviridae (Cammisa-
Parks, Cisar, Kane, & Stollar, 1992). Similar viruses (termed “insect-specific
flaviviruses”; ISFs) were not known to circulate in nature until the identifi-
cation 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. mos-
quitoes (Aedes flavivirus) in Puerto Rico (Cook et al., 2006). A mosquito-
specific 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 cyto-
pathic 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 trans-
ovularilly (virus on egg). Field evidence of ISF-infected immature mosqui-
toes (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
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 mod-
ified 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 arthropod-
borne 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 post-
infection (Beaty et al., 1985). A resistance period for dual infection was iden-
tified 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 dem-
onstrated 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 replica-
tion as well-incompatible interactions between viral proteins exclude repli-
cation 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
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 transmissi-
bility 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 well-
established 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. Specif-
ically, 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, &
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 trans-
mission 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 transmis-
sion 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 occur-
ring 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; Fechter-
Leggett, 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 indi-
cating 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.,
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 rep-
licative 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 vec-
tors 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 vec-
tors 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 evi-
dence 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 mos-
quito passages has demonstrated that portions of the viral genome most com-
monly 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 mos-
quito’s innate immune system to thwart an arboviral infection at lower tem-
peratures. 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,
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 RNA-
silencing 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 subse-
quent 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 regu-
lation 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 suscep-
tibility 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
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 (Apte-
Deshpande, 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 infectiv-
ity 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 stud-
ies 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. quin-
quefasciatus 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 Wolbachia-
positive 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). Fur-
ther 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 undoubt-
edly shed light on the basis of the differential effectiveness of Wolbachia
strains as well on the grounds of the heterotypic effectiveness against differ-

ent arboviral agents. The fact that mosquito culture systems demonstrate no
retardation of viral transcription strongly indicates a posttranscriptional reg-
ulation to be a likely candidature for this inhibitory phenomenon. Never-
theless, 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 illus-
trate 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, dif-
ferent arboviruses continually evolve at the population level due to exposure
to these various selective pressures that are further complicated by transmis-
sion 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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CHAPTER TWO

The Role of Environmental,

Advances in Virus Research, Volume 89 # 2014 Elsevier Inc. 39

environment determines successful transmission of the virus. Once believed to be

pathogen that determine the ability of a given mosquito species to serve as a

3. ORAL VECTOR INFECTION

as the initial barrier determining vector competence (hereto the term

of Aedes aegypti to DENV-2 infection and dissemination (Molina-Cruz

Virus must be taken in an infectious blood meal from a viremic host in

3.1. Viral determinants of infection

infection; however, the reciprocal mutation introduction did not confer

3.2. Receptor-mediated midgut infection

3.3. Vector genetics that modulate viral infection

as 1000-fold for geographic strains of Ae. albopictus infected with CHIKV

3.4. Blood-feeding factors and vector infection

E2 glycoprotein with high-mannose sugars that are poor inducers of type-I

4. MIDGUT ESCAPE AND DISSEMINATION

4.1. Intrahost viral populations

Presumably, these differences in viral diversification are the result of the

solely due to limitations of persisting in a dual-host cycle (Ciota et al., 2009).

4.2. Viral population bottlenecks

4.3. Physiological and pathological changes imparted by

are clear temperature and precipitation thresholds that predict subsequent

6.1. Superinfection exclusion

6.2. Vertical transmission of arboviruses in mosquitoes

Hardy, 1982), Jamestown Canyon virus (Hardy, Eldridge, Reeves,

7. UTILIZING MOSQUITO BIOLOGY TO INHIBIT

2011) gene-silencing pathways (Blair, 2011). In addition to targeting

2013). The RNAi pathway affords a target for arboviruses to antagonize in

strains as well on the grounds of the heterotypic effectiveness against differ-

RNA pathway in anti-viral defense. BMC Microbiology, 11, 45. http://dx.doi.org/

372(1), 176–186. http://dx.doi.org/10.1016/j.virol.2007.10.011, S0042-6822(07)

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