REVIEW

published: 23 January 2019

doi: 10.3389/fmicb.2019.00022

Arbovirus-Mosquito Vector-Host
Interactions and the Impact on
Transmission and Disease
Pathogenesis of Arboviruses
Yan-Jang S. Huang 1,2 , Stephen Higgs 1,2 and Dana L. Vanlandingham 1,2*
1
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS,
United States, 2 Biosecurity Research Institute, Kansas State University, Manhattan, KS, United States

Hundreds of viruses, designated as arboviruses, are transmitted by arthropod vectors

in complex transmission cycles between the virus, vertebrate host, and the vector.
With millions of human and animal infections per year, it is critical to improve our
understanding of the interactions between the biological and environmental factors that
play a critical role in pathogenesis, disease outcomes, and transmission of arboviruses.
This review focuses on mosquito-borne arboviruses and discusses current knowledge
of the factors and underlying mechanisms that influence infection and transmission of
Edited by: arboviruses and discusses critical factors and pathways that can potentially become
Tonya Michelle Colpitts,
Boston University, United States
targets for intervention and therapeutics.
Reviewed by: Keywords: arthropod vectors, arboviruses, vertebrate hosts, infection, transmission
Saravanan Thangamani,
The University of Texas Medical
Branch at Galveston, United States
Stephen Wikel,
ARBOVIRUSES, TRANSMISSION, AND HUMAN DISEASES
Quinnipiac University, United States
The first revelation that mosquitoes can vector human pathogens was the discovery of mosquito
*Correspondence: transmission of filarial worms by Sir Patrick Manson (Manson, 1878). In 1881, Carlos Finlay
Dana L. Vanlandingham
hypothesized that the etiologic agent responsible for yellow fever might be carried by mosquitoes,
[email protected]
and this theory was proven to be true by Major Walter Reed who, in 1900 made the first observation
Specialty section:
that a human virus, yellow fever virus (YFV), can be transmitted by mosquitoes. The concept
This article was submitted to and term of “arthropod-borne” virus transmission was first introduced to the field of virology
Virology, in 1942 (Hammon and Reeves, 1945), and as pioneering discoveries are published, the term
a section of the journal arthropod-borne virus, or arbovirus, continues to evolve. In the publication, “Arthropod-borne
Frontiers in Microbiology and rodent-borne viral diseases”, by the World Health Organization, members of its scientific
Received: 31 October 2018 group defined arboviruses as “viruses that share the characteristic of being naturally maintained
Accepted: 09 January 2019 through biological transmission between susceptible vertebrate hosts by hematophagous arthropods
Published: 23 January 2019 or transovarial transmission from infected female arthropods to her progeny” (WHO, 1985). Whilst
Citation: mosquitoes are responsible for the transmission of many medically important arboviruses, other
Huang Y-JS, Higgs S and arthropod taxa play an important role in vectoring human viruses as well. For example, ticks,
Vanlandingham DL (2019) especially hard ticks under the Ixodidate family, are vectors for several tick-borne flaviviruses such
Arbovirus-Mosquito Vector-Host
as tick-borne encephalitis virus (TBEV), Powassan virus (POWV), and Omsk hemorrhagic fever
Interactions and the Impact on
Transmission and Disease
virus, and bunyaviruses such as Crimean-Congo hemorrhagic fever virus.
Pathogenesis of Arboviruses. Although human-to-human transmission through the exchange of infectious body fluids has
Front. Microbiol. 10:22. been reported for several arboviruses, the majority of arthropod-borne transmission associated
doi: 10.3389/fmicb.2019.00022 with human diseases is achieved by infected arthropods feeding on people. Often, as for example

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Huang et al. Arbovirus-Vector-Host Interactions

in the case of West Nile virus (WNV), the virus is transmitted and subtropical areas (Chang et al., 2007). Temperature also
by mosquitoes to reservoir and amplifying hosts that develop influences the kinetics of replication and dissemination of
a viremia that is sufficient to infect mosquitoes, and humans, viruses in the mosquito and becomes a determinant for vector
as dead-end hosts, become infected with potentially severe competence. Under laboratory conditions, extrinsic incubation
and potentially fatal disease. Under certain circumstances, for period of arboviruses at higher temperatures is often shorter
example when vectors and naive hosts are abundant, an enzootic than at lower temperatures (Liu et al., 2017). In nature, the
cycle limited to a specific region or period may become epizootic fluctuation of temperature has also been found to alter vector
with large numbers of cases. Because of the requirement for the competence of disease vectors (Carrington et al., 2013a,b). As a
virus to replicate in both the arthropod and vertebrate host, it is consequence of environmental influence on the vector, outbreaks
immediately apparent that the process of biological transmission of arboviruses, for example dengue viruses, can be correlated
creates multiple opportunities for interactions among vertebrate with season, specifically those that promote high mosquito
hosts, vectors, and viruses. These interactions can occur on population densities (Campbell et al., 2013). As with the vector,
multiple levels that can ultimately impact transmission patterns the environment can influence distribution, abundance, and
and disease pathogenesis. At the ecological level, transmission even susceptibility of vertebrate hosts. Effects could include
patterns of arboviruses can be influenced by the specific species migration patterns and reproductive status, and availability of
of vector or vertebrate hosts involved in the transmission young naïve hosts. One of the most aggressive dispersal of
cycle. This is particularly true as the geographic distribution of CHIKV in human history was attributed to an abrupt increase
vector species and arboviruses is often expanded through the of Ae. aegypti population followed by emergence of the East-
movements of humans and cargo. This dispersal can lead to Central-South-African genotype of CHIKV in the coastal region
a change in the transmission patterns of arboviruses as they of Kenya (Chretien et al., 2007). As a consequence of the
are introduced into new regions. A particularly good example outbreaks, millions of individuals were infected in the archipelago
is the spread of Aedes aegypti from Africa to the New World along the Indian Ocean between 2005 and 2006. Numerous
several hundred years ago. Ae. aegypti subsequently became attempts have been made to produce predictive models that
responsible for the urban transmission of YFV in the Americas relate environment/climate to disease outbreaks, perhaps the
and continues to be a medically important species following most successful being for Rift Valley fever virus (Linthicum et al.,
the emergence of dengue virus (DENV), chikungunya virus 1999). Environmental influences on arboviral infections and
(CHIKV), and Zika virus in the New World (Tabachnick, 1991; vectors has been reviewed by Mellor (Mellor, 2004). Although it
Gould et al., 2003). Culex quinquefasciatus was also introduced is logical to suggest that if temperatures increase in specific areas,
to the New World, presumably near the same time as Ae. then one could anticipate some redistribution of, for example,
aegypti, and has become an important vector species for WNV. tropical mosquitoes into traditionally temperate zones, and as
The ongoing invasion of Ae. albopictus into various locations a consequence, perhaps a geographic change in the distribution
since the late 20th century has further created significant of associated arboviruses, but to date there seems to be no
ecological and health threats because of its competence for compulsive proof that this has happened (Gould and Higgs,
various arboviruses (Vanlandingham et al., 2016). Although 2009).
the introduction of competent vector species and pathogenic Regardless of the arbovirus or the vector species, a significant
arboviruses into new geographic regions, where immunologically number of arboviral infections in humans are asymptomatic
naïve hosts are present, can profoundly change the epidemiology or cause only a mild transient fever. Arboviruses can also
of arboviruses, the change in epidemiology and overall disease cause other non-specific symptoms which are observed in most
burden often has little impact on disease pathogenesis in viral infections, such as rash and myalgia. Specific clinical
humans. In this review, we focus on the discoveries of manifestations can be generally classified into distinct categories
interactions between arboviruses, vectors, and vertebrate hosts of clinical outcomes: hemorrhagic fever, encephalitis or central
at the cellular and molecular levels, which ultimately changed nervous system involvement, or arthritis (Barrett and Higgs,
transmission patterns and disease pathogenesis. Such discoveries 2007; Higgs, 2008; Higgs and Vanlandingham, 2016). The
have potential applications as targets for antiviral therapies, possibility of further development of severe disease is dependent
vaccines, or preventive interventions for arboviral diseases. on the arbovirus and the physiological and immunological
Of obvious relevance to geographic distribution is the effect condition of the patient. Several groups of arboviruses can cause
of the environment on both the biology of the vectors but also viral hemorrhagic fever including mosquito-borne flaviviruses
the relationships between the vectors and the viruses. Climate, under the YFV and DENV serocomplexes, tick-borne flaviviruses
and particularly temperature and rainfall, have a significant effect such as Omsk hemorrhagic fever virus and Kyasanur Forest
on the distribution and abundance of different mosquito species. disease virus, mosquito-borne bunyaviruses in the genus
At higher temperatures, the mosquito life cycle is shorter than Phlebovirus such as Rift Valley fever virus, and tick-borne
at lower temperatures, and typically there is a species-specific bunyaviruses in the genus Nairovirus such as Crimean-Congo
lower temperature threshold at which the species cannot survive. hemorrhagic fever virus. The hemorrhagic form of the disease is
This in essence determines the geographic distribution of the likely to have the highest public health significance based on the
species. For instance, Ae. aegypti, one of major vectors for case numbers. This is largely due to the increased disease burden
arboviruses, is more sensitive to low temperature in nature than created by DENV, which is the etiological agent for dengue
Ae. albopictus, limiting its geographic distribution to tropical hemorrhagic fever (Gubler, 1998).

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Huang et al. Arbovirus-Vector-Host Interactions

Another important re-emerging mosquito transmitted life-saving technologies, including vaccine candidates and novel
pathogen is YFV, which has caused significant mortality in control strategies.
Africa since 2015 due to a shortage of the global stockpile of
the 17D vaccine (The Lancet, 2016). The movement of viremic Cellular Factors Controlling Arbovirus
YF-infected people from Africa to China has caused concern Infections in Arthropods
that a yellow fever outbreak could occur in the immunologically Identification of intrinsic factors, for example immune-related
naïve populations in Asia where the urban vector, Ae. aegypti, genes that control the infection process of arboviruses in
is present in large numbers (Calisher and Woodall, 2016). arthropods, has long been an important topic for arbovirus
The other important form of severe disease in humans is research (Sim et al., 2014). As demonstrated with vectors
encephalitis and related central nervous system diseases caused collected from different geographic regions, genetic variations
by several arboviruses from different virus families (Higgs and in vector species and populations can often lead to differences
Vanlandingham, 2016). For example, members of flaviviruses in the functionality of protein products that can subsequently
within the Japanese encephalitis virus and TBEV complexes determine susceptibility and vector competence (Gubler et al.,
are known to cause encephalitic diseases in humans (Higgs 1979; Tabachnick et al., 1985). Without access to genome
and Vanlandingham, 2016). Similarly, infection of New World sequences of all medically important species, studies in the past
alphaviruses such as Venezuelan equine encephalitis virus and have relied on various genetic tools such as electrophoretic
Eastern equine encephalitis virus also cause encephalitis in methods and quantitative trait loci mapping to determine the
humans. Orthobunyaviruses, in the California serogroup, such as link between vector genetics and vector competence (Gomez-
La Crosse virus, are etiological agents for pediatric encephalitis, Machorro et al., 2004; Bennett et al., 2005). These pioneering
especially in North America (Vasconcelos and Calisher, 2016). studies and the information obtained from model organisms
Distinct from the viral hemorrhagic fever diseases caused generated fundamental knowledge needed to discover genes and
by systematic infection, lethal encephalitis is normally the pathways that control the process and outcomes of arbovirus
consequence of infection taking place in an incidental host, infections. The development of different molecular biological
which normally does not develop viremia high enough to techniques in the late 20th century provided tools, for example
sustain the transmission cycle but which often succumbs to RNA interference (RNAi), to examine individual factors as
disease (Weaver and Barrett, 2004). The third form of human determinants of the outcome of arbovirus infection in vivo.
disease caused by arbovirus infection is arthritis, which is mainly Despite these advances and the availability of sequence data for
associated with the Old World alphaviruses, for example CHIKV several important species of mosquito, we still do not understand
(Suhrbier et al., 2012; Higgs et al., 2018). Although the disease the basis of species specific susceptibility to virus infection and
is rarely lethal, it often leads to a significant disease burden in vector competence.
the population and substantial economic loss in endemic regions
(Higgs et al., 2018). Genetic Approach to Study Vector Competence and
Vector-Virus Interactions
The effort to determine the role of vector genetics on
VIRUS-VECTOR INTERACTIONS arbovirus susceptibility originally focused on the study of vector
competence of various mosquito populations for arboviruses and
Infection of an arthropod vector is typically required to the impact of selection during mosquito colonization on vector
sustain the transmission cycle of arboviruses. This process competence. By targeting the variable genetic loci encoding
can have a direct impact on the pathological outcome of several metabolic enzymes, the impact on Ae. aegypti genetics
arboviruses in humans or other vertebrate hosts and virus- during the process of colonization was first shown to be
vector interactions can often determine the epidemic potential linked with their susceptibility to YFV (Lorenz et al., 1984).
of arboviruses (Schneider and Higgs, 2008). Approaches This observation was subsequently examined using comparative
used to better understand virus-vector interactions can be studies on the susceptibility of two subspecies of Ae. aegypti, Ae.
multidisciplinary examining vector biology, molecular genetics, aegypti aegypti, and Ae. aegypti formosus, to other flaviviruses,
and arbovirology. Infection with a virus, for example WNV, dengue virus, and yellow fever virus (Tabachnick et al., 1985;
can influence transcription of mosquito genes and subsequent Vazeille-Falcoz et al., 1999). Using a quantitative genetics
protein abundance, for example in salivary glands, and can approach Ae. aegypti formosus, which is restricted to sylvan and
potentially influence virus infection and disease development rural areas of West Africa, was shown to have significantly lower
in the vertebrate host (Girard et al., 2010). Effects of saliva susceptibility to human arboviruses due to genetic differences
on the vertebrate and its influence on virus infection of the from its related competent vector species, Ae. aegypti aegypti
vertebrate is discussed in more detail below. Recently, it has (Bosio et al., 1998). Although this topic continues to be actively
also become evident that the presence of the vector microbiome investigated in other studies involving vector competence of
(symbiotic bacteria) persistently infecting mosquitoes can also different vector populations for urban and sylvatic strains of
alter virus-vector interactions and affect the infection process DENV and YFV (Dickson et al., 2014), a substantial challenge has
of arboviruses (van den Hurk et al., 2012; Dennison et al., emerged as some Ae. aegypti formosus have become domesticated
2014). Knowledge of virus-vector interactions is often helpful in (Brown et al., 2011). For example, in an outbreak caused by
identifying critical factors that assist in the development of new DENV-3 in Cape Verde, an archipelago of 10 islands off the

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Huang et al. Arbovirus-Vector-Host Interactions

Atlantic coast of West Africa, Ae. aegypti formosus was reported responses is less well understood in ticks. Evidence from in vitro
to be a competent vector that is highly susceptible to YFV and studies have shown that replication of Langat virus also triggers
CHIKV, which are both regarded as re-emerging arboviruses the RNAi response in Ixodes scapularis ticks, most likely through
with high public health significance (Vazeille et al., 2013). similar mechanisms as are found in mosquitoes (Schnettler
Despite much effort, and availability of some mosquito genomes, et al., 2014). Despite observations that indicate RNAi plays
the genetic basis of species and population-specific mosquito an important role in arthropod antiviral immunity and the
susceptibility/refractoriness to infection, genetic determinants of initial success in using RNAi to develop control strategies for
dissemination and of transmission are still poorly understood. arboviruses (Franz et al., 2014), the use of RNAi as a control
measure remains limited. For example, a genetically engineered
Innate Immunity of Arthropod Vectors and Arbovirus RNAi response targeting DENV failed to consistently maintain
Infection resistance in transgenic mosquitoes (Franz et al., 2009, 2014).
With evidence indicating that mosquito genetics can influence The future of exploiting the RNAi response to produce pathogen-
susceptibility and competence for arboviruses, the next critical resistant arthropods remains unclear. However, the results thus
step was to identify pathways and specific genes that act as far with RNAi are similar to other findings that mosquitoes
determinants for the infection process. The early search for rely on multiple mechanisms to defend against infection by
determinants of vector competence focused on factors acting as arboviruses. For example, o’nyong-nyong virus replication is
immune components in mosquitoes and largely benefitted from impeded by the heat shock protein 70B in Anopheles gambiae
the pioneering studies conducted on Drosophila melanogaster, (Sim et al., 2007). The Toll, IMD, and Jak-STAT pathways have
a model organism used for studies on innate immunity and been shown to be activated in response to Sindbis virus and
pattern recognition receptors (Hoffmann, 2003). One of the DENV in Ae. aegypti and WNV in Culex pipiens (Xi et al.,
well-characterized pathways discovered was the antiviral RNAi 2008; Luplertlop et al., 2011; Zink et al., 2015). Interestingly,
response that was originally discovered and characterized in fruit while arthropod vectors develop immune responses to limit viral
flies (Galiana-Arnoux et al., 2006). Both fruit flies and mosquitoes infection, arboviruses have also developed what may be regarded
share this conserved mechanism which detects the presence of as immune-escape strategies. This becomes evident by further
RNA viruses by detecting double-stranded RNA which triggers characterization performed with CHIKV and Semliki Forest virus
the RNAi response. The presence of double-stranded RNA that showed infection of alphaviruses results in the suppression
indicates viral replication in infected cells (Westaway et al., 1997; of signaling pathways (Fragkoudis et al., 2008; McFarlane et al.,
Li et al., 2002). The development of an in vivo RNAi mediated 2014).
knockdown system utilizing the Sindbis virus expression system,
in Ae. aegypti mosquitoes, was initially used to knockdown a Genomic and Transcriptomic Tools to Characterize
variety of endogenous mosquito genes and viral genes in vivo. the Vector-Virus Interactions
This system effectively reduced DENV-2 and YFV transmission In addition to studies focused on immune responses, the broader
in Ae. aegypti (Olson et al., 1996; Higgs et al., 1998), reduced picture of how arthropod vectors interact with arboviruses
luciferase expression in transgenic Ae. aegypti (Johnson et al., was further clarified once tools became available to study
1999), and knockdown of a GATA factor which functions as a genomes and transcriptomes. Progress was particularly evident
repressor gene in blood feeding Ae. aegypti mosquitoes (Attardo for studies on medically important mosquito species with
et al., 2003). Utilizing a similar alphavirus based o’nyong-nyong available genomic sequences. Through the characterization of
virus expression system, the importance of RNAi in limiting the transcriptome responses, it became clear that the infection
replication of a medically important arbovirus in mosquitoes was process of arboviruses involves complex responses related to
demonstrated in Anopheles gambiae infected with o’nyong-nyong detoxification, metabolism, immunity, DNA replication, protein
virus (Keene et al., 2004). Additional supportive evidence for the translation, and apoptosis (Sanders et al., 2005; Girard et al.,
importance of RNAi in limiting arbovirus infections was obtained 2010; Tchankouo-Nguetcheu et al., 2010; Colpitts et al., 2011).
through additional experiments using DENV and Sindbis virus Several genes identified in these transcriptome studies have been
in Ae. aegypti (Campbell et al., 2008; Sanchez-Vargas et al., further characterized to determine the importance of arbovirus
2009; Khoo et al., 2010). Interestingly, the antiviral RNAi infections in arthropod vectors. Because of its importance in
response in mosquitoes is also modulated by environmental limiting virus infections in vertebrate hosts, apoptosis signaling
conditions, especially extrinsic temperature. Cooler temperatures pathways were evaluated in a number of studies; however, the
may increase the susceptibility of mosquitoes to arboviruses by role of apoptosis in arbovirus infection in arthropod vectors
impairing the RNAi response (Adelman et al., 2013). remains contradictory (Wang et al., 2008, 2012; O’Neill et al.,
As more data suggests that RNAi can mediate potent antiviral 2015; Eng et al., 2016). This may be due to the fact that approaches
responses in mosquitoes, strategies utilized by arboviruses used to knockdown individual caspases and inhibitors failed
to escape the immune response have also been discovered. to consider the hierarchical organization and complexity of
Through the expression of subgenomic RNA molecules, several apoptotic pathways.
arboviruses have developed an efficient strategy to ensure the In addition to the concern that the experimental approach
ability to replicate by sequestering the antiviral RNAi machinery of manipulating individual genes may not produce conclusive
(Moon et al., 2015). In contrast to the significant advancements and biologically relevant results, there is evidence indicating
made in mosquitoes, the importance of RNAi-mediated antiviral that different components of a physiological response such

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Huang et al. Arbovirus-Vector-Host Interactions

as digestion, nutritional status, and reproductive status may observations indicating that mutations result in different
be involved in both the defense mechanisms of arthropod phenotypes and consequences of infection were reported prior
vectors and also play a critical role in the arboviral life cycle to the genomic era. The earliest example in the literature may
(Wikel et al., 2017). Such complexity is well exemplified by be the effects caused by the serial passage of YFV in chicken
studies showing the ubiquitin proteasome pathway is involved embryos originally used to generate the attenuated 17D strain.
in the arthropod immune response and release of infectious Although the genetic composition was unknown, the attenuated
viruses. By monitoring and manipulating the expression of 17D strain was shown to be deficient in disseminating from
a mosquito ubiquitin protein, Ub3881 in various tissues, its the midgut of infected mosquitoes (Whitman, 1939; Miller
antiviral function was demonstrated by labeling the DENV and Adkins, 1988; McElroy et al., 2008). This phenotype
envelope protein for degradation as an immune response to was later demonstrated to be caused by differences in the
limit viral infection (Troupin et al., 2016). In addition to its genetic composition between the attenuated and virulent strains
role in innate immunity, the ubiquitin proteasome pathway is (McElroy et al., 2005, 2006a,b). The distinct phenotypes between
also critical for the release of DENV particles from an infected virulent and attenuated strains of YFV in mosquitoes provide
midgut of Ae. aegypti (Choy et al., 2015). Although these a unique model to characterize vector-virus interactions. The
studies have identified several cellular factors contributing to the identification of individual determinants in the viral genome
susceptibility and refractoriness of specific arthropod vectors to which control the outcome of infection in mosquitoes has not
arboviruses, it has become clear that vector competence is likely yet been achieved. However, knowledge derived from these
to be determined by multiple pathways in arthropods. While earlier studies has led to several discoveries indicating that
manipulation of individual genetic products remains the only domain III of the flavivirus envelope protein of YFV, and of
feasible approach to characterizing vector-virus interactions, the other flaviviruses, is a critical region for the determination of
broad view on how different pathways and their interactions infectivity in arthropod vectors (Erb et al., 2010; Huang et al.,
can influence vector competence for arboviruses is still needed 2014).
for improved mechanistic understanding of the infection,
dissemination, and transmission processes of arboviruses in Emergence of West Nile Virus Genotypes and Its
arthropod vectors. Public Health Significance in North America
Since the introduction of WNV into the United States in
Viral Genetics and Arbovirus Infections 1999, the study of viral genetics and its influence on virus-
in Arthropod Vectors vector interaction in nature has been extensively carried out
Whilst the manipulation of individual genes in arthropod by genotypic and phenotypic analyses (Mann et al., 2013). The
vectors is helpful for identifying genes and pathways that first significant increase in its clinical incidence and public
are involved in the arboviral infection process, the other health significance was attributed to the emergence of the North
aspect of vector-virus interactions is the virus. Virus mutants America/WN 2002 genotype. This genotype efficiently displaced
with distinct phenotypes have been used to infect arthropods the New York 1999 genotype with two major surges in the
in order to identify the relationship between viral sequence numbers of human infections with neurological diseases in 2002–
and phenotype (McElroy et al., 2005; Anderson and Rico- 2003 and 2012. Its selective advantage was later determined to
Hesse, 2006; Kenney et al., 2012). This approach has led to be the reduced extrinsic incubation period in C. pipiens and
substantial advancements in the field of arbovirology due to C. tarsalis but not the dosage required for the establishment
the low fidelity during the replication of the RNA genome of infection in mosquitoes (Ebel et al., 2004; Moudy et al.,
among medically important arboviruses in the families of 2007; Vanlandingham et al., 2008). While phylogenetic analyses
Flaviviridae, Togaviridae, and Bunyaviridae leading to rapid suggest the change of phenotype is linked to the single V159A
viral genetic changes (Xia et al., 2016). In nature, the substitution in the envelope protein (Davis et al., 2005), direct
evolution of arboviruses has repeatedly resulted in changes in experimental evidence, using a reverse genetics approach to
epidemic potential and transmission patterns (Davis et al., 2005; characterize the substitution, is still missing to test the hypothesis
Schuffenecker et al., 2006; Schuh et al., 2014). Several of these that the gain of selective advantage in mosquitoes is due to
examples were found to be associated with increased infectivity the single mutation. In the last 20 years, primarily because of
or transmission efficiency in arthropods which becomes the the availability and use of molecular methods, especially those
mechanism for the emergence and re-emergence of arboviruses based on Polymerase Chain Reactions (PCR), it has become
(Moudy et al., 2007; Dubrulle et al., 2009). Characterization possible to detect viruses in mosquitoes that were not revealed
of viral mutants, either isolated from nature or derived from using traditional methods because they killed neither cell cultures
laboratory experiments, can aid in the characterization of nor suckling mice. A comprehensive review of these arthropod-
arboviral genotypes that can alter the phenotype of arboviruses specific viruses that do not infect vertebrates and are maintained
in arthropod vectors. by transovarial transmission was recently published (Calisher
and Higgs, 2018). A question that has been asked is whether
Yellow Fever Virus as a Model to Study Viral Genetics or not these viruses may influence vector infection with other
and Its Impact on Vector-Flavivirus Interactions closely related viruses. Potential interference with WNV was
Although the techniques of determining genetic sequences evaluated by Goenaga et al. (2015) and Hall-Mendelin et al.
of arboviruses did not become available until the 1980s, (2016).

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The Relationship Between Chikungunya Virus Odhiambo et al., 2014). The finding demonstrates that the
Mutation and Vector Competence structure genes encoded in the M segment are likely to contain
Another example that viral genetics contributes to the epidemic the determinants for vector range, infectivity, and efficiency of
potential of an arbovirus was reported in the re-emergence of developing the disseminated form of infection in arthropods, as
CHIKV in the Indian Ocean in 2005 (Huang et al., 2018). previously reported over 30 years ago (Beaty et al., 1981, 1982;
Historically, CHIKV is known to be transmitted by Ae. aegypti Sundin et al., 1987).
in its endemic regions, especially Africa and Southeast Asia. The mechanisms controlling virus genetic reassortment are
The change of its primary vector species to Ae. albopictus not completely understood. Characterization of the dynamics
was reported in an outbreak on several islands in the Indian of reassortment has been investigated using mosquitoes fed
Ocean (Reiter et al., 2006). With its rapid dispersal in the two La Crosse virus strains in order to induce homotypic
late 20th century, the species was also subsequently shown reassortment (Beaty et al., 1985). Interestingly, the incidence
to trigger local transmission of CHIKV in Italy and France of dual infection and presumably the subsequent genetic
(Angelini et al., 2007; Gould et al., 2010). Although mutations reassortment significantly increases if two viruses are ingested
subsequently detected in other regions of its genome may simultaneously or separately with an interval less than 48 h
contribute to this process, the adaptation of CHIKV to Ae. (Beaty et al., 1985). Although the conclusion from the
albopictus was primarily driven by the acquisition of the A226V La Crosse virus dual infection studies are different from
mutation in its E1 protein (Tsetsarkin et al., 2007; Tsetsarkin and another superinfection model based on dual infections of
Weaver, 2011). Surprisingly, despite the fact that the selective the alphavirus, CHIKV, and the flavivirus, DENV (Nuckols
advantage in transmission created by the E1-A226V mutation et al., 2015), these studies demonstrate that superinfection of
in the East-Central-South-African genotype is maintained in multiple arboviruses is likely to occur through the ingestion of
several epidemics recorded in different geographic regions (de viremic blood meals. Although the dynamics of superinfection
Lamballerie et al., 2008), the introduction of CHIKV into the New has been previously studied, another important question that
World in 2014 was caused by the dispersal of its Asian genotype, remains unaddressed is the packaging mechanisms of the three
which lacks the A226V mutation. As the introduction and genomic segments to generate reassortant viruses. It is generally
autochthonous transmission of the East-Central-South-African believed that the packaging of the three genomic segments
genotype was detected in Brazil in 2015 (Nunes et al., 2015), it is not a random process and requires complex interactions
is unclear if the A226V mutation will be selected through the between viral RNA and nucleoproteins in order to generate
transmission by Ae. albopictus in the New World. a reassortant bunyavirus (Pringle et al., 1984; Hornak et al.,
2016).
Genetic Reassortment of Bunyaviruses and
Vector-Virus Interactions
In contrast to genetic drift created by accumulation of individual Infection With Symbiotic Bacteria and
mutations in viruses containing single-stranded RNA genomes, Vector-Virus Interactions
genetic shift is attributed to the reassortment of segmented The role of symbiotic bacteria in persistently infected mosquitoes
genomes (Briese et al., 2013). Segment reassortment can has become increasingly studied and understood in vector-
potentially cause a large number of genetic changes in an virus interactions (Wikel, 1996; Higgs, 2004). There have been
arbovirus. Among the medically important arboviruses, the several species of symbiotic bacteria that have been found to
process of genetic reassortment is unique to viruses in the family interfere with the infection of arboviruses in arthropod vectors.
Bunyaviridae (Briese et al., 2013). Segment reassortment can A particularly good example is the infection of Wolbachia in
occur if either the vertebrate host or the arthropod vector is mosquitoes that can lead to a decrease in vector competence
simultaneously infected with two or more related viruses. In for arboviruses (van den Hurk et al., 2012). Persistent infection
nature, reassortant bunyaviruses have been previously isolated of Wolbachia has been reported in all arthropod species except
from both vertebrate hosts and arthropod vectors (Klimas et al., for Ae. aegypti. Species of Wolbachia have been evaluated
1981; Reese et al., 2008). This process has been reported to for their potential to suppress the vector population due to
contribute to the emergence of new and more virulent viruses. the discovery that artificially infected Ae. aegypti can lead
A reassortment event in 1997 and 1998, between Bunyamwera to cytoplasmic incompatibility and embryonic lethality when
virus and Batai virus (BATV) resulted in the emergence of Ngari mated with uninfected Ae. aegypti in nature (Hoffmann et al.,
virus (NRIV), a reassortant orthobunyavirus, which caused a 2011). Wolbachia-based vector control strategies have been
large viral hemorrhagic fever outbreak in several East African shown to suppress the replication of WNV in its vector,
countries, including Kenya and Somalia (Gerrard et al., 2004; C. quinquefasciatus (Glaser and Meola, 2010). Other studies
Briese et al., 2006). NRIV contains the S and L segments of have also demonstrated its interference in the replication and
Bunyamwera virus and the M segment of BATV. Because the transmission of DENV, CHIKV, and Zika virus in Ae. aegypti
M segment encodes the structural genes of BATV, the virion (Walker et al., 2011; van den Hurk et al., 2012; Aliota et al.,
of NRIV is likely to share similar structure and biochemical 2016); however, the bacteria did not show any significant
properties of BATV. NRIV was found to infect Anopheles species reduction of vector competence of Ae. aegypti for YFV and
mosquitoes which are also the likely vector species for BATV C. tarsalis for WNV (van den Hurk et al., 2012; Dodson et al.,
(Huhtamo et al., 2013; Ochieng et al., 2013; Liu et al., 2014; 2014).

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Huang et al. Arbovirus-Vector-Host Interactions

Several mechanisms have been proposed to explain the mosquitoes. Therefore, such interactions have become important
resistance to arboviruses induced by Wolbachia infection. The factors in determining the transmission patterns and disease
majority of available evidence suggests Wolbachia infection pathogenesis of arboviruses. This is discussed in more detail
leads to the activation of immune responses and limits below.
viral replication (Terradas and McGraw, 2017). However,
mechanistic evidence regarding the pathways and effector
genes is still needed (Bian et al., 2010; Pan et al., 2012; Roles of Arthropod Saliva in Disease
Rances et al., 2012, 2013). With its success in limiting Pathogenesis
arbovirus replication in Ae. aegypti, it is no surprise that A recent review by Higgs et al. (2017), describes mosquito
this approach was further evaluated as a control strategy for modulation of arbovirus-host interaction, whilst Higgs and
Ae. albopictus (Moretti et al., 2018), a highly invasive vector Vanlandingham (2016) compare and contrast ticks and
species competent for various arboviruses. Although initial mosquitoes with an emphasis on tick-borne transmission.
evidence suggested that Ae. albopictus, artificially infected by Although many of the proteins present in mosquito saliva have
an Wolbachia strain derived from Drosophila under laboratory unknown functions, there are others that are known to alter
conditions, can interfere with infection of CHIKV and DENV normal physiological functions of the host which facilitates
(Blagrove et al., 2012, 2013), infection of Wolbachia among the acquisition of blood meals. The experimental transmission
Ae. albopictus in nature did not lead to any significant of arboviruses in animal models has shown that saliva leads
impairment of infection and dissemination of CHIKV (Ahmad to more severe disease than virus inoculation without saliva
et al., 2017). As Wolbachia infection triggers a different set of (Schneider and Higgs, 2008). Functional characterization
physiological responses in Ae. albopictus, particularly immune has demonstrated multiple roles of saliva in vasodilation,
responses, further evaluation is needed before a comprehensive pain suppression, anti-inflammation, anti-coagulation, and
understanding of what impact Wolbachia infection has on anti-hemostasis. Pathogen transmission with saliva has also
infection of arboviruses in Ae. albopictus (Molloy and Sinkins, been found to alter host immune responses. These functions
2015). often are associated with the increase of viral replication and
In contrast to the impact of the artificial introduction of dissemination in vertebrate hosts and the augmentation of
Wolbachia into medically important mosquito species, another pathological outcomes mainly by compromising the host
approach focuses on characterizing the interactions among immune mechanisms.
arthropod vectors, arboviruses, and symbiotic bacteria. This type In several in vitro experimental systems, the growth of
of interaction has been shown to be particularly important to arboviruses can be promoted by the addition of salivary
the susceptibility of mosquitoes to arboviruses. For example, the components or their extracts, which suppress the production
colonization of microbiota in the midgut of Ae. aegypti triggers of antiviral cytokines (Fuchsberger et al., 1995; Hajnicka et al.,
basal immune responses that suppress the infection of DENV 1998; Limesand et al., 2003). Although the cell lines chosen in
(Ramirez et al., 2012). Although the majority of the bacterial these studies may not necessarily reflect the tissue tropisms of
population in a mosquito midgut and their interactions remains arboviruses, it has become evident that the immunomodulation
to be investigated, it has already become clear that specific species caused by arthropod saliva is critical for disease pathogenesis
may have an inhibitory effect, not only to arboviruses, but also to of different arboviruses. In spite of the differences in the
other pathogenic microorganisms (Ramirez et al., 2014). feeding processes between ticks and mosquitoes, the presence of
salivary gland components from both species has been shown
to cause enhancement in disease severity of several arboviruses
belonging to different virus families in different animal models
VECTOR-HOST INTERACTIONS AND (Dessens and Nuttall, 1998; Edwards et al., 1998; Limesand
THEIR IMPACT ON TRANSMISSION AND et al., 2000; Schneider et al., 2006; Hermance and Thangamani,
DISEASE PATHOGENESIS OF 2015). While the species of vertebrate hosts are different in
ARBOVIRUSES various studies, the immunomodulation properties of saliva from
mosquitoes generally promote the TH 2 immune response and
Arbovirus infections are established through the feeding prevents the clearance of arboviruses and other intracellular
of arthropod vectors on vertebrate hosts, a much more pathogens transmitted by arthropods, which is mainly driven
complex process than the simple inoculation of arboviruses by the TH 1 immune response (Kovar et al., 2002; Schneider
using, for example, needle inoculation. This natural feeding et al., 2004; Skallova et al., 2008; Thangamani et al., 2010; Cox
process creates complicated interactions between arthropod et al., 2012). However, the induction of the TH 2 response is
vectors and vertebrate hosts as arboviruses are injected less apparent in the feeding of I. scapularis ticks infected by
through the saliva, which quickly elicits responses to the POWV, indicating the fundamental difference in the saliva-
feeding process from the vertebrate hosts. Depending on the mediated immunomodulation between mosquitoes and ticks
duration of feeding and quantities of arboviruses delivered, (Hermance and Thangamani, 2014). Using WNV as a model,
non-viremic transmission of arboviruses among co-feeding the immunomodulation by mosquito saliva can also be shown
arthropod vectors can also be created in at least two by the change in the recruitment process of T lymphocytes
types of medically important arthropods, namely ticks and to the site of infection and ultimately modulate the antigen

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Huang et al. Arbovirus-Vector-Host Interactions

presentation cell signaling critical for the dissemination of by salivary protein components of arthropods. For example, the
arboviruses (Schneider et al., 2010). The increased recruitment significant increase of DENV viremic titers is associated with the
of neutrophils and monocytes and antigen presentation cell reduced level of cytokines related to adaptive immune responses
migration to draining lymph nodes can also be induced when caused by aegyptin, an Ae. aegypti salivary protein (McCracken
mosquito salivary gland extracts and DENV were delivered et al., 2014). In addition to the impairment of immune responses,
through needle inoculation (Schmid et al., 2016). In addition other novel pathways have also been suggested. Through the
to the change in the local tissues and lymph nodes, the secretion of a serine protease molecule, mosquito saliva has
effect induced by the salivary gland components was also been shown to promote the infection of DENV by increasing
demonstrated to be systemic. When challenge with Rift Valley binding affinity to cellular receptors such as proteoglycan
fever virus was performed in the presence of salivary gland molecules and the induction of cell migration, which can
extract, significant increase in viral load of blood, liver, and potentially facilitate the dissemination of virus (Conway et al.,
brain was observed (Le Coupanec et al., 2013). Whilst the 2014).
feeding process of mosquitoes and ticks are remarkably different, With our increased understanding of the importance of
there has been a significant level of similarity reported in the salivary proteins in the enhancement of arbovirus infection,
immunomodulation caused by tick and mosquito saliva. The it has become apparent that the salivary components of
stimulation in the cell recruitment process by tick saliva has also arthropods are critical for the pathogenesis of arboviruses.
been found to be important for disease pathogenesis of tick- Such knowledge may later become helpful in the development
borne arboviruses. During the prolonged feeding period of ticks, of animal models for disease research and strategies for
phagocytes and neutrophils were reported to be the primary intervention and prevention of severe diseases. However,
cell types recruited to the feeding sites in the very early stage our readers must keep in mind that the results must
of feeding from POWV-infected ticks. Interestingly, the same be interpreted with caution as the modulation of immune
study also indicated that macrophages and fibroblasts are likely responses and enhancement of infection are often observed
to be the primary cell types infected by POWV during the in laboratory animals that are not natural hosts for most
early stage, although the establishment of infection coincided arboviruses.
with the activation of pro-inflammatory cytokines (Hermance
et al., 2016). Interestingly, in spite of the similarity in the
stimulation of cell recruitment process by mosquito and tick Vector-Host Interactions and Arbovirus
saliva, changes in the cytokine expression profile induced by Transmission
tick saliva did not reveal the preferential change toward the In addition to the enhancement of arbovirus infection,
TH 2 response. The production of pro-inflammatory cytokines interactions between arthropod vectors and vertebrate hosts
was triggered in mice exposed to ticks infected by POWV can also be deciding factors for transmission (Higgs and
(Hermance and Thangamani, 2014). The observation may be Vanlandingham, 2016; Higgs et al., 2017). As the vectors
a reflection of the fundamental difference in the cellular and inject infectious viruses into vertebrate hosts, the process
molecular interactions triggered by saliva between mosquito transiently creates a unique environment that contains
and tick feeding. Such knowledge may be critical for our high concentrations of arboviruses and can promote an
understanding of the establishment of infection and disease alternative but important mode of transmission called
pathogenesis of mosquito-borne and tick-borne arboviruses in non-viremic transmission. Such a process occurs when
vertebrate hosts. feeding of infected and susceptible arthropod vectors takes
With the increased understanding of the dynamics of place simultaneously in close proximity to each other on
immunomodulation caused by arthropod saliva, several studies a vertebrate host. The term non-viremic transmission was
have been designed to determine the specific functions of initially used to describe this mode of transmission because
individual salivary components. Targeting the innate and of the lack of detectable viremia in a vertebrate host, and
adaptive immune responses, there have been several known can also be referred to as non-replicative transmission. The
mechanisms that cause the enhancement of arbovirus infection process promotes the transmission of some arboviruses as
in the presence of arthropod saliva. Type-I interferon (IFN) and it may not necessarily require a viremic vertebrate host.
its downstream signaling pathways, one of the most important Through the creation of a local environment that contains
mechanisms in innate antiviral immunity, can be impaired at a high viral load, this mode of transmission can happen in
different levels by salivary proteins. Several molecules in the vertebrate hosts that are resistant or not permissive for viral
saliva of Ae. aegypti have been shown to suppress the type- replication to develop a viremia of a magnitude that exceeds the
I IFN signaling pathway in vitro by suppressing the mRNA threshold titer that is generally assumed necessary to facilitate
expression of type-I IFN, IFN-responsive element, and effector transmission.
genes (Surasombatpattana et al., 2014). Similar suppression Non-viremic transmission was described in Thogoto virus,
of type-I IFN was also observed with the treatment of tick an orthomyxovirus transmitted by the African brown ear tick
salivary gland extract with detailed mechanisms to be determined Rhipicephalus appendiculatus in a guinea pig model (Jones et al.,
(Hajnicka et al., 1998). In contrast to the shutdown of innate 1987). Whilst guinea pigs normally do not develop high viremic
immune signaling that mediates broad-spectrum antimicrobial titers to sustain Thogoto virus transmission, transmission was
immunity, adaptive immune responses can also be compromised found to occur through co-feeding of infected adults with

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Huang et al. Arbovirus-Vector-Host Interactions

uninfected adults or nymphs on the same guinea pig without transmission is likely to be higher when arthropod vectors
detectable viremic titers. As ticks often feed for a prolonged are co-feeding simultaneously in close proximity. However,
period on vertebrate hosts, it is no surprise that this type transmission of WNV can also be achieved with temporal and
of transmission can significantly increase during a longer co- spatial segregation (McGee et al., 2007). The finding further
feeding process which enables longer exposure to infectious confirms the importance of this mode of transmission for
viruses (Jones and Nuttall, 1989). R. appendiculatus has been the transmission of mosquito-borne viruses as it allows the
shown to be more efficient than Amblyomma variegatum as transmission to occur in the sequential feeding of donor and
donor ticks in mediating non-viremic transmission. This finding recipient mosquitoes.
indicates that non-viremic transmission may take place during
co-feeding of certain tick species at higher incidence (Jones
et al., 1990). The characterization of non-viremic transmission CONCLUSION
was quickly expanded to the characterization of transmission
and maintenance mechanisms for TBEV, a zoonotic flavivirus Although our understanding of the interactions among
that is maintained in wild animals, especially rodent species arboviruses, arthropod vectors, and vertebrate hosts continues
(Labuda et al., 1993a,b). Interestingly, in addition to non- to improve, it is apparent that arthropod vectors do not merely
viremic transmission taking place in vertebrate hosts resistant serve as “mobile syringes” to deliver arboviruses to susceptible
to TBEV infection, non-viremic transmission can also have vertebrate hosts. Arthropods vectors, which actively mount
significant influence on the dynamics of TBEV transmission immune and anti-viral responses to limit arbovirus infections,
and evolution in susceptible vertebrate hosts. Non-viremic can provide a unique intracellular environment to select and
transmission through co-feeding I. ricinus has a significantly promote the transmission of specific viral populations which
higher incidence in vertebrate hosts that are susceptible to can ultimately change the transmission patterns of arboviruses.
TBEV (Labuda et al., 1993c). More importantly, this mode of Genetic engineering to enhance vector immune or anti-viral
transmission can also occur in vertebrate hosts which have responses, or manipulation of the vector microbiome to reduce
immunity against TBEV (Labuda et al., 1997). The finding vector competence or suppress vector populations are all being
suggests non-viremic transmission can take place for a longer considered as options to reduce the incidence of vector-borne
period of time and be independent of viremia. It has several viruses. Genetic approaches to modulate viral determinants
important impacts on the evolution and spread of tick-borne of infection, dissemination, and transmission may help in the
arboviruses. This process can result in the selection of avirulent rationale design of safe and efficacious vaccine candidates that
strains with delayed onset of viremic phase and low mortality in cannot be transmitted by arthropods. When feeding on vertebrate
vertebrate hosts. Non-viremic transmission is also advantageous hosts, vector-host interactions have been proven to play a critical
for viral transmission because it allows ticks to complete the role in disease pathogenesis and further create alternative routes
engorgement process. This hypothesis is also supported by of transmission. Understanding these interactions have been
the finding that genetic elements of the European subtype shown to provide unique strategies for the development of
of TBEV promote non-viremic transmission by showing a animal models for disease pathogenesis. We can certainly expect
higher incidence of non-viremic transmission in I. ricinus than that as our understanding continues to grow, new technologies
the Siberian subtype (Khasnatinov et al., 2016). However, a will be developed. Combined with other findings in the field,
significant gap of knowledge is the undetermined virulence a better understanding of these unique interactions will likely
of both subtypes in vertebrate hosts as natural reservoirs. An contribute to the formulation of more novel control strategies
overview of non-viremic and salivary-activated transmission of in the future. Applications of the relatively recently developed
tick-borne viruses is provided by Higgs and Vanlandingham CRISPR technology, for example, not only offers a new method
(2016). for genetic manipulation, but furthermore may be used as a
Whilst non-replicative or non-viremic transmission was gene drive to establish desirable traits such as resistance to
mainly observed through the prolonged co-feeding of ticks, infection in vector populations (National Academies of Sciences,
it was later demonstrated that non-viremic transmission can 2016).
also take place in mosquito-borne arboviruses (Huang et al.,
2017). Secretion of WNV at sufficiently high concentration
by infected donor mosquitoes was shown to result in non- AUTHOR CONTRIBUTIONS
viremic transmission to uninfected recipient mosquitoes (Higgs
Y-JH, SH, and DV drafted and edited the article together.
et al., 2005). This process relies on a transient non-propagative
environment with high viral load at the sites where co-feeding
of multiple arthropods takes place (Reisen et al., 2007). Because FUNDING
the increased temporal and spatial separation in the feeding
of donor and recipient arthropods is likely to reduce the This research received funding from the State of Kansas National
availability of infectious viruses, the efficiency of non-viremic Bio and Agro-defense Facility Transition Fund.

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Huang et al. Arbovirus-Vector-Host Interactions

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