Critical Reviews™ in Eukaryotic Gene Expression, 27(4):331–340 (2017)

Genetically Modified Aedes aegypti to Control

Dengue: A Review
Muhammad Qsim,a Usman Ali Ashfaq,a,* Muhammad Zubair Yousaf,b Muhammad Shareef
Masoud,a Ijaz Rasul,a Namrah Noor,a & Azfar Hussainc
Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad-38000,
a

Pakistan; bCentres of Excellence in Science & Applied Technologies, Islamabad, Pakistan; cBuffalo Research Institute
Pattoki, Pakistan

*Address all correspondence to: Usman Ali Ashfaq, Department of Bioinformatics and Biotechnology, Government College University Faisalabad,
Pakistan; Tel./Fax: +92 03314728790, E-mail: usmancemb@gmail.com

ABSTRACT: Dengue is an acute infectious disease of viral etiology characterized by lymphadenopathy, leucopenia,
headache, biphasic fever, pain in various parts of the body, rashes, and extreme physical weakness. It is a vector-borne
disease caused by a positive-stranded RNA virus of the family Flaviviridae, genus Flavivirus. Dengue inflicts a sig-
nificant health, economic, and social burden on populations of endemic areas. Dengue virus is transmitted to humans
by the mosquito vector Aedes aegypti. Vaccines against dengue viruses have been claimed to be developed, but as yet
no effective treatment is available. Alternative therapeutic strategies to overcome this disease and its spread are direly
needed. A traditional sterile insect technique (SIT) harms the health of male insects, leading to their reduced ability to
compete for wild-type female insects for breeding. Oxitec (Abingdon, UK) has developed genetically modified (GM)
strains of A. aegypti via the release of insects carrying a dominant lethal (RIDL) strategy. RIDL male mosquitoes offer
a resolution to many of the limitations of traditional SIT, which has resulted in reduced application of SIT in mosqui-
toes. The technique using RIDL mosquitoes is considered to be ecologically friendly and specific. Homing endonu-
clease genes, also called selfish genes, can also be used in genetic modification methods in such a way that the vector
population and its competency can be reduced. GM mosquitoes carrying a gene that transcribes RNA interference can
also be crucial to control expression of RNA viruses. The RNA virus interference pathway is one of the most critical
components of the innate immune system of insects that can frustrate a variety of RNA viruses such as Flaviviruses.
Here, we summarize and focus on alternative techniques used to control dengue spread.

KEY WORDS: RNA interference, CRISPR-Cas9, Aedes aegypti, dengue, sterile insect technique

I. INTRODUCTION response in approximately 7 d. Secondary infection

Dengue, probably the most important arthropod-
borne viral (arbovirus) disease in terms of human
morbidity and mortality, is a major public health
problem worldwide, with ~ 2.5 billion individuals
infected, leading to nearly 25,000 deaths per year.1,2
Clinical signs and symptoms can vary from acute
fever with headache, chills, and muscle or joint pain
to severe life-threatening symptoms. It is estimated
that more than 100 countries with populated of
~ 50–100 million individuals are influenced by this
infection, with Asia, Central and South America,
and Africa the main areas affected.3–5 Dengue infec-
tions may be categorized as primary or secondary.
Primary infection causes an acute fever known as
dengue fever that can be cured by a complex immune
is more severe and can cause dengue hemorrhagic
fever or dengue shock syndrome (DSS).
Dengue, a member of the family Flaviviridae, is
a positive-sense RNA virus. Dengue virus has four
major serotypes (DENV1–4) that are antigenically
distinct from one another, but all cause disease.6,7
Dengue virus is 50-nm-containing lipid mem-
brane, with 180 identical copies of envelop (E) pro-
tein present at the surface of the viral membrane’s
short transmembrane segment. The virus genome
(of about 11,000 bases) is translated into a single
large polyprotein that is cleaved into three structural
and seven nonstructural genes. Short noncoding re-
gions are also present on both the 5′ and 3′ ends.
Structural proteins include capsid (C), E glycopro-
tein, and membrane (M). Nonstructural proteins are

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332
classed as NS1, NS2A, NS2B, NS3, NS4A, NS4B,
and NS5 (Fig. 1).8,9 Structural proteins play a vital
part in the formational organization of virus and
viral entrance into host cells, whereas nonstructural
proteins are key in viral replication and other cel-
lular functions.3,4
No specific vaccine or therapeutic drug is avail-
able yet for dengue. The Sanofi Pasture (Lyon,
France) vaccine chimeric yellow fever virus-DENV
tetravalent dengue vaccine (CYD-TDV) is the most
recent vaccine in clinical trials now. This vaccine,
formed by mixing all four serotype recombinant vi-
ruses, exhibited good results in initial trials, but its
overall efficacy is low, especially against dengue
virus serotype 2 (DENV2).10 A study in India demon-
strated the development of a vaccine for the preven-
tion of dengue fever via the yeast Pichia pastoris.
Noninfectious dengue-virus-like particles were cre-
ated to activate virus-specific immunity. Animal trials
using this vaccine yielded good results, but it is not
yet commercially available.11 There is thus a strong
need to find alternative ways to control this disease.
Vector-borne diseases are key threats to human
health and pose a dreadful and intolerable public
health load on society. To a great extent, developed
FIG. 1: Genome of the dengue virus
Qsim et al.
countries have escaped the morbidity and mortality
that mosquitoes and their arthropod associates inflict
on humans via diseases such as dengue and malaria.
A worldwide increase of vectors is one of the major
reasons for the emergence of different diseases in
new areas. Therefore, there is an increasing demand
to develop strategies for vector control. Since 1950s,
dengue has expanded geographically worldwide at a
high rate. The rise in dengue spread is alarming and
is responsible for many casualties.12 Several factors
are involved in the spread of dengue fever, and the
Aedes aegypti mosquito vector is one of them.10 The
Aedes mosquito is thus a good target for overcoming
dengue fever, but comprehensive work is required to
control this vector.
Because dengue can be controlled by reduction or
elimination of the vector population, the prevention
of dengue virus transmission relies heavily on vector
control. Traditional methods of vector control such
as bed nets and space spraying are largely ineffective
against this daytime biting mosquito, so new strate-
gies are needed. Vector control strategies are classed
into two basic methods: population suppression (re-
duce) or population elimination (replace), both with
the aid of genetically modified (GM) mosquitoes
Critical Reviews™ in Eukaryotic Gene Expression
GM Aedes aegypti to Control Dengue
(GMMs).13 In this review, we focus on several inno-
vative techniques for controlling the dengue vector.
II. SELF-LIMITING POPULATION-
SUPPRESSION MECHANISMS
A. Sterile Insect Technique
The sterile insect technique (SIT) is a species-spe-
cific, environmental friendly method of biological
insect control that is based on the release of large
numbers of sterile insects into the environment.
With the population-suppression method, the vector
number is reduced to minimize disease transmission.
In population replacement, the wild-type vector is
replaced with a GM vector (GMV) that is incapable
of transmitting the disease.14 For this method, a ge-
netic control system is devised based on the release
of radiation-sterilized insects. This technique en-
compasses mating of male GMMs with native fe-
males, resulting in decreased female reproductive
potential.15 Wild female pairing to a sterile male pro-
duces fewer or no children, so the population tends
to decrease. If enough sterile insects are released for
an appropriate period, the target population is con-
trolled or even eradicated locally.16 Large numbers
of sterile males released into the environment mate
with wild-type females, reducing the reproductive
capacity of mosquitoes.17 It is noteworthy that re-
duction of mating competitiveness and sterile fe-
males do not reduce the population, and the fertility
of irradiated males is a residual effect that limits the
approaches to mosquito control using SIT. With SIT,
liberated male mosquitoes do not take blood meals,
resulting in a density-dependent reduction due to le-
thality performance at very early stages, leading to
larval death.18,19 The reduction in fecundity declines
in both arthropod-borne pathogen transmission as
well as the insect population.
Numerous sterilizing techniques for mosquito
control such as chemosterilization, ionizing radiation,
cytoplasmic incompatibility, and chromosome trans-
location have been in use since the 1970s.20 SIT has
been used successfully for more than 50 yr to control
an entire area and/or eliminate several important ag-
ricultural pests and disease vectors, including the re-
gional elimination of the Mediterranean fruit fly and
Volume 27, Issue 4, 2017
333
New World screwworm fly in the United States.21
Although a series of tests were conducted in 1970s
with some success, no SIT programs are currently
in large-scale operation against mosquito species.22
A. aegypti, a mosquito species fit for mass rearing,
appears to be reasonably uniform across large areas,
without the problems of subspecies and barriers to
mating.23 Recently, to predict effectiveness of SIT,
various studies focused on designing mathematical
models to simulate the application of the technique
to A. aegypti.24
1. SIT Advantages versus Traditional
Strategies
• SIT is species specific, thus targeted species
populations only are eliminated.
• The mating behavior of male insects is highly
efficient.
• Wild insect density is condensed, making it
more effective.25
2. SIT Limitations
• A loss of competitiveness mating for wild-type
insects occurs, due to sterilization by irradiation.
• The success of SIT is highly dependent on
release of male mosquitoes only; sterile fe-
males bite and consequently transmit disease
(male mosquitoes do not bite). Therefore, SIT
involves labor-intensive gender-based sep­
aration of male-only populations.
• Irradiation of pupae appears to harm insects;
irradiation as adults is less harmful but oper­
ation­ally much more difficult. In some trials,
sterilizing chemicals were used, such as thio­
tepa, which was effective for sterilization but
led to trace contamination with this mutagenic
chemical.
• Unlike agricultural pests against which the
main SIT programs are directed at present,
mosquito populations can be regulated pri-
marily by density-dependent effects in which
a very fruitful population is maintained at a
level stable for limited resource constraints.
• Fitness costs and operational difficulty of ir-
radiation in SIT field operations.5,6,11
334
To overcome these SIT limitations, GM female
mosquitoes may be used. In female transgenic mos-
quitoes, OX3604C genes are present to produce the
flightless female-tetracycline-repressible pheno-
type. Thus, flightless females become incapable of
mating (Fig. 2).26
B. Release of Insects Carrying a Dominant
Lethal Gene
Population studies using SIT restricted mosquito
control due to the somatic damage, producing an as-
sociated performance reduction in the sterile insects
that inevitably accompanies radiation-based steril-
ization.15 However, modern genetics can overcome
this problem by inserting in insects an engineered
self-limiting gene that eliminates the need for ra-
diation sterilization in a similar SIT program. The
release of insects carrying a dominant lethal (RIDL)
strategy reduces these limitations by way of the re-
combinant DNA technology that was introduced by
Oxitec (Abington, UK). RIDL uses GM male and
female mosquitoes.27,28 OX513A RIDL bisex is a
GM strain from which only males are released to
mate with wild females. The offspring of these mat-
ings die due to late larvae or pupae. Thus, the target
population can easily reduce.16,27 At the same time,
some or all of the descendants of GM mosquitoes
die because of inheriting one or more dominant le-
thal mutations, resulting in the tendency for the pop-
ulation to decline.28,29
FIG. 2: The GM flightless female lethal gene is inserted into the vector (GM male) that breeds with wild-type females
to produce flightless females
Qsim et al.
1. Construction of GMMs
GM male mosquitoes have been developed that con-
tain the lethal gene to combat the spread of dengue
fever. LA513 is a dependent piggyBac-based trans-
poson containing 8.4 kb of nucleotides. Inserting the
LA513 transposon within A. aegypti mosquitoes by
genetic engineering techniques has been performed
to produce toxins in the mosquitoes during the larval
stage under normal conditions. This then causes the
death of larvae. Transposon LA513 mainly consists of
DsRed2 and tetracycline-repressible transcriptional
activator (tTAV) genes. OX513A individuals can be
detected under fluorescent light due to the expression
of a second, dominantly inherited, introduced gene that
encodes for red fluorescent protein (DsRed2). tTAV is
controlled by its binding site tetO, the minimal pro-
moter from Drosophila heat-shock protein (Hsp70),
and the 3′ untranslated region (UTR) sequence from
the Drosophila gene Dmelfs(1)K10. tTAV binds with
its binding site tetO, executing a high-level expression
of tTAV that is toxic under normal conditions. In the
presence of tetracycline, tTAV binds with tetracycline
instead of tetO, leading to the reduced expression of
tTAV. Therefore, this construction gives a very high-
level expression of tTAV in the absence of tetracycline
but executes minimal to no expression in the presence
of tetracycline. The high-level expression of tTAV may
be due to the interaction with the VP16 domain tran-
scription factor, which provides a key to the building
of this deadly system (Fig. 3).29,30
Critical Reviews™ in Eukaryotic Gene Expression
GM Aedes aegypti to Control Dengue 335

Fluorescent Marker

FIG. 3: LA513 transposon structure and function. LA513 is an 8.4-kb piggyBac-based transposon. DsRed is the
fluorescent marker that is used to identify the transgenic gene. The Act5C promoter is used to drive the expression of
DsRed. tTAV is a tetracycline-repressible transcription activator that is controlled by its binding site tetO, the minimal
promoter of Drosophila Hsp70, and a 3’ UTR sequence of Drosophila fs(1)K10 that all control tTAV.

When GMMs are released into the environment
and mate with wild-type females, their offspring in-
herit this trait lethality and die before reaching the age
of maturity (during the larval stage), resulting in a de-
crease of the local mosquito swarms.26 As described
above, the RIDL strategy uses the GM A. aegypti
OX513 abisexual strain (Fig. 4).29 Furthermore, by
means of GM male A. aegypti, reduction or elimina-
tion of the mosquito population of that species can
minimize transmission of the dengue virus to humans.
C. Homing Endonuclease Genes
Homing endonuclease genes (HEGs) were first
discovered in bacteria.6 For insect control, HEGs
provide a powerful mechanism for driving genes
through populations, but although they are pri-
marily used as gene drivers, self-limiting forms of
HEG control are also possible.31 HEGs encode en-
donuclease enzymes that recognize the site, cleave a
20–30-bp sequence present on the chromosome, and
insert HEGs into this site.32 HEGs could be used to
dislocate genes that are essential to pathogen spread
or activate homologous recombination and gene al-
teration to increase the presence of transgenic an-
tipathogen genes.6
Although HEGs are naturally present in mi-
crobes, those that are genetically engineered can
perform functions in mosquitoes.33 Endonucleases
enzymes that recognize the site present only once
in the genome, cleave a 20–30-bp sequence, and in-
serted HEGs are then saved from their own activity.34
HEGs are inserted in their own recognition site, and
recombination repair machinery then uses the HEG-
enclosing strand as a template. Thus, another HEG-
enclosing strand is formed and the number of HEGs
increases. In this manner, HEGs serve as the vehicle
for driving genes through a population.
HEGs can also be planned to identify specific
sequences of mosquito genes required for vector
proficiency.32 On the other hand, HEGs can be used
for population suppression by pointing genes to en-
courage infertility and decrease survival or sex ratio
alterations. So far, HEGs have been positively engi-
neered into A. aegypti.26
III. POPULATION REPLACEMENT FOR
CONTROLLING THE DENGUE VECTOR
Population-replacement techniques involve the in-
sertion of additional or foreign genes to prevent
mosquitoes from transmitting disease. These tech-
niques may include inserting an antipathogen gene,
immune system up-regulator, or a combination of
transgenic approaches that will ultimately result in
the reduction or elimination of disease transmission
from mosquito to human. To assess whether a mos-
quito has reduced disease transmission capability,
the virus titer in the experimental transgenic mos-
quito’s midgut and salivary glands can be measured
and compared to a wild-type control.35,36
At least three genetic-transformation systems
have been described and used successfully in A.
aegypti to generate GMVs. These transformation

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 336

FIG. 4: RIDL mechanism. The LA513 transposon inserts in the produced cultured cell GM larvae, which is kept in the presence of tetracycline to prohibit
gene expression. Eggs are produced after breeding. Lethal genes are expressed and the larvae die due to the absence of tetracycline; wild-type male
progeny are not formed.

Critical Reviews™ in Eukaryotic Gene Expression

Qsim et al.
GM Aedes aegypti to Control Dengue
systems are based on the class II transposable el-
ements (TEs) Mariner (Mos1), Hermes, and pig-
gyBac. Mos1 and piggyBac are the most commonly
used TEs for generating GMVs.37
A. Control through Gene Manipulation
via RNA Interference
RNA interference (RNAi) refers to a gene silencing
mechanism through which double-stranded (ds)
RNA is delivered to cells or organisms. Introduction
of dsRNA into invertebrates can be achieved by ex-
ogenous feeding or injection to create a significant
silencing phenotype.31 Use of the RNAi strategy
(Fig. 5) against the recurrence of dengue virus can
operate effectively in mosquitoes and mosquito
cells. The first evidence in this regard occurred
when the recombinant Sindbis virus (SINV, a sin-
gle-stranded RNA virus) that had a DENV2–pre-
membrane protein (prM) sequence was transformed
into adult female mosquitoes. During replication,
FIG. 5: RNAi mechanism. dsRNA enters the cell and degrades by dicer, resulting in the production of siRNA.
Afterward, the RISC complex binds with siRNA to target and degrade the messenger RNA.
Volume 27, Issue 4, 2017
337
SINV was converted into dsRNA intermediary, ex-
pressing DENV2-prM to activate the RNAi mecha-
nism. The combination of DENV2 and recombinant
SINV with the DENV2-prM sequence reduced
DENV replication in mosquitoes.38
In a mosquito cell-culture expression of a
dsRNA, its hairpin structure is a strong catalyst for
the RNAi mechanism. To make the hairpin struc-
ture, 567 nucleotides of the prM sequence from
DENV2 represents the sense orientation, and the
first 290 nucleotides repeat in an antisense orienta-
tion. DENV2-prM is cloned into plasmid, and after
transcription, DENV2-specific dsRNA is formed,
which halts DENV2 replication. Transfection of
C6/36 (Aedes albopictus) with dsRNA of the ar-
bovirus genome is performed, showing that Aedes
species have an RNAi mechanism that is similar to
other organisms.35 Blair et al. and Sánchez-Vargas
and colleagues demonstrated that RNAi has an im-
portant role in the reduction of dengue infection.39,40
These authors found that adeno-associated virus
338
small interfering (si)RNA infected the dendritic
cells and decreased dengue infection in humans.
Mosquito cell line culture (C6/36, A. albopictus) is
transformed by way of plasmid that contains the in-
verted sequence of the dengue prM gene and forms
290-bp dsRNA. These transformed cells enhance re-
sistance to DENV2. Hence, infection of transformed
cells with DENV2 shows decreased numbers of viral
RNA. These transformed mosquito cells contain all
the machinery of RNAi. This finding proved that
RNAi has an important role in reducing DENV2.41
Transposons (jumping genes) that contain the in-
verted repeat sequence of the prM gene of dengue
virus are transformed in a mosquito’s embryo, along
with the carboxypeptidase A promoter. With that
promoter in place, mosquitoes express the 578-bp
dsRNA product after they imbibe a bloodmeal. When
GMM Carb 77 is attached to DENV2 after a blood
meal, dsRNA is expressed along with viral replica-
tion, thus preventing viral spread from the vector to
the host via the salivary glands. In this way, the RNAi
mechanism accounts for the resistance of DENV2.36,39
The siRNA-mediated silencing of receptors
and facility of clathrin-mediated endocytosis pre-
vent entry of the dengue virus. The proliferation of
HepG2 cells reduces the virus and inhibits dengue
fever from developing into serious stages. Specified
cellular genes are involved in endocytosis opera-
tions and dynamics of the cell structure, an important
task of infection DENV. siRNA-directing genes are
linked with clathrin-mediated endocytosis.39
Villegas-Rosales et al.42 projected that three
siRNAs have ability to silence the four DENV se-
rotype genomes by pointing out NS4B and NS5 se-
quences. siRNA along with RNAi self-processing
machinery have a role in the preclusion of unadorned
dengue infection. In the development of new thera-
peutic drugs, RNAi can play an important part.37,39
RNAi-mediated gene silencing assays in A. ae-
gypti cells reveal the role of Unk7703 and sterol car-
rier protein 2 (SCP2) in DENV restriction and host
factor, respectively. Unk7703 is conserved in Aedes
culex and Anopheles and is involved in cell signaling
pathways. Similarly, SCP2 facilitates cholesterol
uptake in A. aegypti cells. Knockdown or chemical
inhibition of SCP2 effectively inhibits DENV repli-
cation in engineered A. aegypti Aag2 cells.43
Qsim et al.
B. Genome Engineering with CRISPR/Cas9
A relatively new approach to the gene-drive system
widely used nowadays for dengue vector control is
CRISPR-associated Cas9 gene editing. Clustered
regularly interspaced palindromic repeats (CRISPR)
and CRISPR-associated (Cas) genes are compo-
nents of an adaptive immune system that are found
in a wide variety of bacteria and archaea.44
1. Mechanism
The CRISPR-Cas9–based gene-drive system works
by induction of a dsDNA break (DSB) at a tar­get
site. The chief components of the CRISPR-Cas9
system are (1) a synthetic single-guide RNA (sgRNA),
which is a small RNA containing 17–20 bases of
complementarity to a specific genomic sequence,
and (2) the Cas9 nuclease derived from Strep­to­­­
coccus pyogenes (SpCas9). The complex of SpCas9
and sgRNA induces a DSB at sequences of the ge-
nome that are directly 5′ to a protospacer-adjacent
motif (PAM) and are complementary to the sgRNA
recognition site. The PAM sequence for SpCas9 is
NGG, which occurs approximately once every 17 bp
in the A. aegypti genome, making it possible to target
essentially any locus.45 Injection of CRISPR-Cas9
reagents for genome editing at the preblastoderm-
stage embryo results in germ-line mutant pheno-
types. DNA repair in a homology-dependent manner
using the Cas9 system leads to a net increase in copy
number. Thus, generation of a loss-of-function mu-
tant allele by site-directed mutagenesis in a heritable
manner is a key feature of gene-drive studies for
vector control.46
IV. CONCLUSION
Dengue is a mosquito-borne disease caused by any
one of four closely related dengue viruses (DENV1–
4). Classic dengue fever, also known as break bone
fever, is characterized by an acute onset of high
fever. Frontal headache, retro-orbital pain, pain in
muscles and joints, anorexia, nausea, hemorrhagic man­
ifestations, rash, and leucopenia are the major symp-
toms of the disease. The A. aegypti mosquito is the
main vector that transmits viruses that cause dengue.
Critical Reviews™ in Eukaryotic Gene Expression
GM Aedes aegypti to Control Dengue
A. aegypti belongs to the Flaviviridae family and
contains different structural and nonstructural pro-
teins. The severity of dengue fever leads to hemor-
rhagic fever and eventually DSS. Millions of people
around the globe are affected every year by dengue
fever. Currently, no commercial vaccine is available
against the dengue virus.
A combination of sterile insect technique (SIT)
and genetic engineering approach (RIDL) can be used
to reduce the dengue vector population and overcome
the limitations of traditional SIT. With the advent
of modern genetics, approaches including the use of
HEGs, RNAi, and CRISPR-Cas9 show promising re-
sults in vector control. However, large-scale produc-
tion and field study of these techniques is still required.
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