Medical and Veterinary Entomology (2019), doi: 10.1111/mve.

12376

Understanding the mechanism of Chikungunya virus

vector competence in three species of mosquitoes
A. G H O S H 1 , T. M U L L A P U D I 1 , S. B O M A N N A 1 , B. K. T Y A G I 2 ,
V. R A V I 1 and A. D E S A I 1
1
Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, Karnataka, India
and 2 Centre for Research in Medical Entomology, Madurai, Tamil Nadu, India

Abstract. Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp.

mosquitoes. The present study investigated vector competence for CHIKV in Aedes
aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role
of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes
spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV
DRDE-06. Infection of midguts and dissemination to heads was confirmed by
immunofluorescence assay at different time points. A plaque assay was performed
from mosquito homogenates at different time points to study CHIKV replication. Pres-
ence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was
detected by virus overlay protein binding assay (VOPBA). The identity of these pro-
teins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae.
albopictus midguts and dissemination to heads was observed to be similar. A plaque
assay performed with infected mosquito homogenates revealed that CHIKV replication
dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA per-
formed with mosquito midgut membrane proteins revealed that prohibitin could serve
as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of
CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the
non-permissiveness of these mosquitoes to infection.
Key words. Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, chikungunya,
interacting protein, mosquito, vector competence.

Introduction gene (E1-A226V) of CHIKV, which led to the recognition of Ae.

Chikungunya virus (CHIKV) is an arbovirus belonging to the
Togaviridae family and is transmitted to humans by the bite
of infected mosquitoes. Aedes aegypti and Aedes albopictus
have been implicated as the primary vectors for CHIKV (Higgs
& Vanlandingham, 2015). Although CHIKV was first isolated
in Calcutta in 1963 (Ravi, 2006), this virus received global
attention as a re-emerging virus after the massive 2005–06
outbreak on the La Reunion Island, which perpetually invaded
the Indian subcontinent. Thereafter, chikungunya has continued
to be endemic in India. Phylogenetic studies reported that
re-emergence event was facilitated by a mutation in the envelope
albopictus as a new vector in addition to the existing Ae. aegypti
(Schuffenecker et al., 2006; Tsetsarkin et al., 2007).
At a national perspective, both Ae. aegypti and Ae. albopictus
mosquitoes are prevalent throughout the country (Kalra et al.,
1997; Eapen et al., 2010). Additionally, both strains of CHIKV,
A226 (Gurav et al., 2012; Afreen et al., 2014; Kumar et al.,
2014; Parashar et al., 2015; Naresh et al., 2016) and the mutant
A226V (Santhosh et al., 2009; Kumar et al., 2012; Ray et al.,
2012) are known to co-circulate causing epidemics in India.
Several studies conducted outside India have investigated the
vector competence of Ae. aegypti and Ae. albopictus mosquitoes
for CHIKV A226 (Girod et al., 2011; Vega-Rúa et al., 2014;

Correspondence: Anita Desai, Department of Neurovirology, NIMHANS, Bangalore 560029, Karnataka, India. Tel.: +91 080 26995778. E-mail:
anitasdesai@gmail.com

© 2019 The Royal Entomological Society 1

2 A. Ghosh et al.
Chen et al., 2015; Vega-Rúa et al., 2015). However, barring a
single study on Ae. aegypti mosquitoes (Gokhale et al., 2015),
there are no reports from India that have investigated the vector
competence of Ae. albopictus mosquitoes for CHIKV A226.
The present study therefore aimed to determine CHIKV vector
competence in laboratory reared Ae. aegypti and Ae. albopictus
mosquitoes in Madurai, South India, for their (i) comparative
efficiency in supporting CHIKV replication and (ii) the molecu-
lar basis of competence in terms of presence of virus-interacting
proteins on the mosquito gut epithelium facilitating CHIKV
entry into cells. Culex quinquefasciatus mosquitoes, the vector
for West Nile virus, are also found in India (Sudeep et al., 2015).
Because Cx. quinquefasciatus mosquitoes are generally incom-
petent with respect to transmitting CHIKV (Jupp et al., 1981;
Richards et al., 2010; van den Hurk et al., 2010), this species
served as a non-permissive control vector throughout the study.
Materials and methods
Mosquitoes
Mosquito rearing and oral infection was performed at the
Centre for Research in Medical Entomology (CRME), Madu-
rai, Tamil Nadu. Eggs of Ae. aegypti and Cx. quinquefasciatus
mosquitoes were obtained from mosquito colonies maintained
at CRME. Aedes albopictus eggs were obtained from colonies
maintained at the field station, Virudhachalam, Cuddalore dis-
trict, Tamil Nadu. F2–F5 generation mosquitoes were used in
the present study.
Mosquito rearing
Eggs were collected from Aedes and Culex spp. and allowed
to hatch in deoxygenated (1.2 parts per million) water in a quart
Mason jar at 27 ∘ C. The larvae were fed on 1 : 1 mixture of
powdered yeast and dog food to complete larval development,
which varied from 7 days to 23 days. Pupae were then separated
from the larvae and placed in the adult cage. The adults were
fed by means of cotton pledget soaked in a mixture containing
packed duck red blood cells and 10% sucrose, placed in the cage.
A temperature of 27 ± 1 ∘ C with 70–80% relative humidity was
maintained in the mosquito colony.
Oral infection of mosquitoes
Oral infection of mosquitoes was carried out in adult female
mosquitoes (7 days post emergence) as described previously
(Pesko et al., 2009; Tchankouo-Nguetcheu et al., 2010).
CHIKV DRDE-06, which does not contain the A226V muta-
tion (Kumar et al., 2014), was used for all experiments. A
complete infectious blood meal for mosquitoes was prepared by
mixing 1 mL of packed duck red blood cells + 1 mL of CHIKV
infectious cell culture fluid (108 pfu/mL) + 0.5 mL FBS and
20% sucrose. The blood mixture was pre-warmed to 37 ∘ C using
a water bath for 5 min. For the experiment, 3–5-day-old female
© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
mosquitoes were released into 15 × 15 × 15 inch cages and
pre-starved of sucrose feed for 2–26 h. In a biosafety cabinet,
cotton pledgets were soaked in the blood meal mixture and
then placed in the Petri dish and introduced into the mosquito
cage. Approximately, 200 μL of the infectious blood meal was
aliquoted separately and used to back-titre the sample. The
blood meal titre of CHIKV was calculated to be 6 × 105 pfu/mL.
Mosquitoes were allowed to feed for 45–60 min. Thereafter,
only those mosquitoes that had taken a blood meal (identified
by fully engorged abdomen) were separated into secondary
containment cage. These mosquitoes were then maintained on
10% sucrose solution at 28 ± 10 ∘ C with 80% relative humidity
until completion of the experiment.
To obtain uninfected controls, mosquitoes of each species
were fed with a blood meal without CHIKV in the same manner.
Those with an engorged abdomen post blood meal were used as
uninfected controls.
Mosquitoes were anaesthetized at defined time points and dis-
sected to obtain midguts and heads. Midgut and head squashes
were prepared separately on clean glass slides and fixed with 4%
paraformaldehyde for 1 h at room temperature. Slides were then
washed thrice with phosphate-buffered saline (PBS) for 5 min
each followed by rinsing twice in 100% methanol. Slides con-
taining squashes were preserved in 100% ethanol at −20 ∘ C.
CHIKV susceptibility of mosquito midguts and dissemination
to heads
CHIKV infection of mosquito midgut was confirmed by per-
forming an immunofluorescence assay (IFA) with midguts
squashes on clean glass slides until 14 days post infec-
tion (PI). Midgut squashes of five mosquitoes from each
species were studied at each time point. Midgut squashes from
uninfected control mosquitoes (five mosquitoes per species
at each time point) were included as negative controls. Slides
stored at −20 ∘ C in ethanol previously were re-equilibrated
in graded series of ethanol (75% < 50% < 25%) followed
by re-equilibration in sterile PBS. The specimens were per-
meabilized in 0.2% Triton X-100 for 1 h at room temperature.
Slides were washed again with PBS (3 × 5 min) and blocked
for 30 min with PBS containing 5% bovine serum albumin.
The slides were drained and incubated overnight at 4 ∘ C
with 1 : 100 anti-eastern equine encephalitis virus monoclonal
antibody (catalogue number MAB8754; Chemicon International
Inc., Temecula, CA, U.S.A.), which exhibits cross-reactivity
with CHIKV envelope (Reddy et al., 2012). Further washing
was performed with PBS (3 × 5 min) on a shaker. The midguts
were incubated with 1 : 200 fluorescein isothiocyanate tagged
secondary conjugate (catalogue number 621120380011730;
GeNei, Merck, Bangalore, India) for 1 h at room temperature
in a humid chamber. Slides were again washed with PBS and
observed under a fluorescence microscope (Nikon, Tokyo,
Japan).
Dissemination of CHIKV to mosquito heads was studied by
IFA on dissected heads and midgut squashes at: 4, 8, 12, 16, 20,
24 and 28 days PI. Five mosquitoes from each species were used
for preparing head and abdominal squashes for each time-point.

Midgut and head squashes from control uninfected mosquitoes
for each time point were included as negative controls.
Preparation of mosquito homogenates for plaque assay
and CHIKV RNA extraction
Mosquito samples were processed to obtain homogenates to be
used for CHIKV RNA extraction and a plaque assay as described
previously (Pesko et al., 2009). Five mosquitoes from each
species at each time point were aliquoted in 500 μL of BA-1 dilu-
ent (1% bovine serum albumin, 0.6% Tris, pH 7.6, 7.5% sodium
bicarbonate, 100 U/mL per 100 μg/mL penicillin/streptomycin,
1 μg/mL; Fungizone; Bristol-Myers Squibb, Chester, U.K.) and
stored at −70 ∘ C. Mosquito tissues were homogenized and then
centrifuged at 3220 g for 4 min at 4 ∘ C. The supernatant obtained
was passed through a 0.2pμm filter and used for the plaque assay.
The plaque assay was performed in triplicate using Vero cells on
a single homogenate prepared from five mosquitoes per species
at each time point. Dilutions (10-fold) of mosquito homogenates
were added to separate wells (200 μL/well). Appropriate con-
trols were included at each time point.
Whole mosquito tissue-extract supernatant previously
obtained at 4, 8, 12, 16, 20, 24 and 28 days PI was also
used for CHIKV RNA extraction in accordance with the manu-
facturer’s instruction (QIAamp Viral RNA Mini kit; catalogue
number 52906; Qiagen, Hilden, Germany). RNA was extracted
from 100 μL of the diluted mosquito tissue extract (diluted
1: 10000). A one-step real-time reverse transcriptase poly-
merase chain reaction (RT-PCR) was performed using CHIKV
specific primers and probes coding for the non-structural pro-
tein 4 (nsP4; genome position 6856–6981) gene as described
previously (Lanciotti et al., 2007).
CHIKV 6856F: 5’-TCA CTC CCT GTT GGA CTT GAT
AGA-3’.
CHIKV 6981R: 5’-TTG ACG AAC AGA GTT AGG AAC
ATA CC-3’.
PROBE1: 5’-FAM-AGG TAC GCG CTT CAA GTT CGG
CG-BHQ-1-3’.
The total volume of mastermix composition for CHIKV gene
detection was 25 μL, consisting of 12.5 μL of 2 × PCR reaction
buffer (Applied Biosystems, Foster City, CA, U.S.A.), 100 μm
primers, 1 μL of enzyme mix, 15 μm probe, 3.6 μL of nuclease
free water and 7.5 μL of RNA template. The cycling conditions
were 50 ∘ C for 30 min, 94 ∘ C for 10 min followed by 45 cycles
at 95 ∘ C for 15 s and 60∘ C for 1 min. All samples were tested
in triplicates. CHIKV RNA isolated from infectious cell culture
supernatant was used as positive control. Uninfected mosquito
homogenate was also included as a control.
Preparation of mosquito midgut brush border membrane
fraction (BBMF) proteins
Mosquito midgut BBMF proteins were isolated from adult
female mosquito midgut epithelial cells as reported previ-
ously (Paingankar et al., 2010). Midguts were dissected from
adult female mosquitoes (five mosquitoes per species) under
a binocular microscope and the peritrophic membranes and
malpighian tubules were removed. The midguts were rinsed
© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
Understanding chikungunya virus vector competence 3
in ice cold buffer A (0.3 m mannitol, 5 mm EGTA, 20 mm
Tris/HCl, 2 mm phenylmethylsulfonyl fluoride) and stored at
−80∘ C until use.
Midguts were homogenized in buffer B (0.3 m mannitol, 5 mm
EGTA, 20 mm Tris/HCl, 2 mm phenylmethylsulfonyl fluoride,
2 mm Triton X-100) and allowed to stand on ice for 20 min
and then centrifuged at 2000 g for 15 min at 4∘ C to remove
membranous organelles. The supernatant was collected and
kept on ice. The pellets were re-extracted in buffer B. The
supernatant from both extractions was centrifuged at 23 200 g
for 60 min at 4 ∘ C. The resulting pellet was then re-suspended in
buffer A and protein concentration was quantified by NanoDrop
(2000/2000c; ThermoFisher Scientific, Waltham, MA, U.S.A.).
Virus overlay protein binding assay (VOPBA)
VOPBA of the membrane proteins was carried out
as described previously (Das et al., 2009). BBMF pro-
teins (50 μg) was resolved by 10% sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by western blotting (100 mA constant) onto a nitro-
cellulose membrane (Bio-Rad, Hercules, CA, U.S.A.). The
membrane was blocked overnight using 5% skimmed milk
powder followed by washing thrice (5 min each) with PBST
(PBS + 0.05% Tween 20). Subsequently, the nitrocellulose
membrane was incubated with infectious CHIKV cell culture
supernatant (titre 106 ) for 3 h at room temperature with gentle
rocking. After another round of washing with PBST, the mem-
brane was incubated with anti-CHIKV polyclonal antibody
(catalogue number 04-0008; IBT Bioservices, Gaithersburg
Maryland) for 2 h at room temperature. Secondary incubation
was carried out with anti-rabbit biotinylated antibody (cata-
logue number AP182B, Millipore, Billerica, MA, U.S.A.) for
1 h at room temperature. The membrane was further incubated
with streptavidin peroxidase conjugate for 45 min at room
temperature and washed thoroughly with PBST. To confirm
the specificity of the assay, a no-virus control was included
where the membrane was not overlaid with CHIKV cell cul-
ture supernatant. Additionally, the membrane fractions used
were subjected western blotting using antibodies to GAPDH
(catalogue number PA1-987; ThermoFisher Scientific), which
is abundant in eukaryotic tissue fractions and has been used
as a loading control in previously reported studies (Arik et al.,
2014).
The BBMF membrane protein bands interacting with CHIKV
were visualized by using a commercial chemiluminiscence kit
(catalogue number 34580; SuperSignal West Pico Chemilumi-
nescent Substrate; Thermo Scientific, USA) in accordance with
the manufacturer’s instructions.
CHIKV-interacting protein band(s) for Ae. aegypti and Ae.
albopictus midgut proteins were excised from Coomassie
Brilliant Blue stained gel and submitted to the Institute of
Bioinformatics (India) for liquid chromatography-mass spec-
trometry (LC-MS) analysis (Shevchenko et al., 2006). Mass
spectrometry analysis of samples was performed in a LTQ
Orbitrap XL™ ETD Hybrid Ion Trap-Orbitrap mass spec-
trometer (ThermoFisher Scientific). The data thus generated
was searched using the homology search tool sequest
4 A. Ghosh et al.

Fig. 1. Chikungunya virus (CHIKV) infection of mosquito midguts by an immunofluorescence assay (IFA). Smears were prepared from midgut
squashes of five mosquitoes per species at each time point, orally infected with CHIKV. All five Aedes aegypti and Aedes albopictus mosquitoes at each
time point (100%; 5/5) were positive for CHIKV antigen (apple green fluorescence). None of the five Culex quinquefasciatus and uninfected control
mosquitoes (0%; 0/5) showed the presence of CHIKV antigen at any time point. A representative image from one mosquito per species is shown. The
fluorescence intensity observed is represented as ‘+’ (low fluorescence), ‘++’ (moderate fluorescence) and ‘+++’ (high fluorescence). [Colour figure
can be viewed at wileyonlinelibrary.com].

(http://proteomicswiki.com/wiki/index.php/SEQUEST) against IFA was performed on midgut squashes prepared from

VectorBase (https://www.vectorbase.org).
Statistical analysis
To compare the replication kinetics, a Mann–Whitney
U-test was carried out to find out the level of significance
between CHIKV titres obtained at different time points for Ae.
aegypti, Ae. albopictus and Cx. quinquefasciatus mosquitoes
by plaque assay using software available online (https://
www.socscistatistics.com/tests/mannwhitney). P < 0.05 was
considered statistically significant.
Results
Confirmation of CHIKV infection of mosquito midguts
and localization by IFA
In the present study, hundred female mosquitoes per species
were released into cages to provide them with an infectious
blood meal. Mosquitoes that had taken the infectious blood
meal were identified by their fully blood engorged abdomen.
Approximately 70% of mosquitoes from a colony introduced
for feeding were found to have engorged abdomens upon
individual examination.
CHIKV infected Ae. aegypti, Ae. albopictus and Cx. quinque-
fasciatus mosquitoes at 2, 6 and 14 days PI. The detection of
CHIKV antigen was confirmed by the presence of apple green
fluorescence in the midgut squashes of Ae. aegypti and Ae.
albopictus mosquitoes at all the time points in all (100%) of the
mosquitoes tested (5/5). No fluorescence was observed in Cx.
quinquefasciatus mosquitoes (0%; 0/5) and uninfected control
mosquito midgut squashes (Fig. 1) at any time point. The inten-
sity of fluorescence signifying the presence of CHIKV antigen
was the least (+) at 2 days PI, improved at 6 days PI (++) and
maximum at 14 days PI (+++) suggesting significant infection
occurred in both Ae. aegypti and Ae. albopictus (Fig. 1).
Furthermore, examination of individual CHIKV infected Ae.
aegypti and Ae. albopictus midguts to investigate virus antigen
localization revealed that the virus followed two different pat-
terns in these two species (Fig. 2). In Ae. aegypti, CHIKV anti-
gen localization in infected midguts was sparse in the ante-
rior and posterior regions at 2 days PI. The virus was then
observed to infect the entire midgut as time progressed. Exami-
nation of infected Ae. albopictus mosquito midguts revealed that
CHIKV infected the entire midgut 2 days PI (Fig. 2). Further
time points showed that the viral antigen was detected through-
out the midgut until the last time point studied (14 days PI).
The presence of CHIKV antigen was observed at all the time
points in all (100%) the Aedes sp. mosquitoes tested (5/5). The

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
 Understanding chikungunya virus vector competence 5

Fig. 2. Localization of Chikungunya virus (CHIKV) in infected Aedes sp. mosquito midguts by an immunofluorescence assay. (A) Aedes aegypti
midgut squashes. (B) Aedes albopictus midgut squashes. CHIKV initially infects the anterior and posterior regions of Ae. aegypti midguts (white
arrows) and then progressively infects the entire midgut. CHIKV infection was observed in the entire midgut of Ae. albopictus from 2 days PI and not
localized to any specific region. All five Ae. aegypti and Ae. albopictus mosquitoes at each time point (100%; 5/5) were positive for CHIKV antigen
(apple green fluorescence). The fluorescence intensity observed is represented as ‘+’ (low fluorescence), ‘++’ (moderate fluorescence) and ‘+++’ (high
fluorescence). [Colour figure can be viewed at wileyonlinelibrary.com].

intensity of fluorescence signifying the presence of CHIKV anti-
gen was the least (+) at 2D and 4 days PI, improved at 6 days
PI (++) and was the maximum at 10 and 14 days PI (+++)
(Fig. 2).
Dissemination of CHIKV in Aedes sp. mosquitoes
The appearance of CHIKV antigen in mosquito heads after
infecting midguts was detected by IFA performed on squashes
prepared from midguts and heads separately at 4, 8, 12, 16,
20, 24 and 28 days PI. In case of Ae. aegypti mosquitoes,
CHIKV antigen was first detected only in the midgut squashes
prepared from infected mosquitoes at 4 days PI. CHIKV antigen
was simultaneously observed in midgut and head squashes of
infected Ae. aegypti mosquitoes at 8 days PI. Thereafter, midgut
and head squashes were positive for CHIKV antigen by IFA
at all time points studied (until 28 days PI) (Fig. 3). In the
case of Ae. albopictus mosquitoes, the viral antigen was first
observed in infected midgut squashes at 4 days PI (Fig. 3),
whereas the antigen was first observed in head squashes at
8 days PI (Fig. 3). Thereafter, CHIKV antigen was concurrently
observed in midgut and head squashes of infected Ae. albopictus
mosquitoes from 8 days PI onwards until the last time point
studied (28 days PI) (Fig. 3). To summarize, the presence of
CHIKV antigen was observed at all the time points in all
(100%) the Aedes sp. mosquito midguts tested (5/5) and smears
prepared from heads 8 days onwards (100%; 5/5). CHIKV
antigen could not be detected in smears prepared from heads
of Aedes sp mosquitoes prior to 8 days (0%, 0/5). Midguts
of Cx. quinquefasciatus mosquitoes and control uninfected
mosquitoes did not show the presence of viral antigen (0%;
0/5) at any time point during the study (Fig. 3). In the case
of Aedes sp. mosquito midguts, the intensity of fluorescence
denoting CHIKV infection in midguts was low (+) at 4 days
PI, moderate (++) at 8 days PI and highest (+++) at 12 days PI
and subsequent time points (Fig. 3). Additionally, in Aedes sp.
mosquito heads, the fluorescence intensity was low at 8 days PI,
moderate at 12 days PI and attained highest intensity at 16 days
PI and all subsequent time points (Fig. 3).
Quantification of infectious CHIKV from mosquito
homogenates by plaque assay
Plaque assay was performed to quantitate infectious CHIKV
obtained from all three mosquito homogenates at 4, 8, 12, 16, 20,
24 and 28 days PI. The mean titres of infectious virions obtained
from triplicate experiments at each time point from Ae. aegypti,
Ae. albopictus and Cx. quinquefasciatus mosquito homogenates
are shown in Table 1.
In the case of Ae. aegypti, the highest titre of infectious CHIKV
(9.5 × 105 pfu/mL) was recovered at 16 days PI, whereas, in the
case of Ae. albopictus mosquitoes, the highest titres of infectious
virions (13.5 × 105 pfu/mL) were observed at 20 and 24 days PI,

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
6 A. Ghosh et al.

Fig. 3. Chikungunya virus (CHIKV) infection of mosquito midguts and subsequent dissemination to heads. (A) The observations of the
immunofluorescence assay experiment. (A-I) Aedes aegypti specimens. (A-II) Aedes albopictus specimens. (A-III) Culex quinquefasciatus specimens.
CHIKV antigen (apple green fluorescence) was observed in Ae. aegypti and Ae. albopictus mosquito midgut (MG) squashes 4 days post infection (PI)
onwards in all five mosquitoes tested at all time points (100%; 5/5). The viral antigen was also seen in all five infected mosquito head (H) squashes of
both species 8 days PI onwards (100%; 5/5). Note that CHIKV antigen was not detected in any of the five midgut squashes of Culex quinquefasciatus
mosquitoes that had taken an infectious blood meal (0%; 0/5) and control mosquitoes at any time point. (B) Fluorescence intensity indicating the level
of virus infection in terms of ‘+’ (low fluorescence), ‘++’ (moderate fluorescence) and ‘+++’ (high fluorescence) at different time points. [Colour
figure can be viewed at wileyonlinelibrary.com].

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376

Table 1. Tabular representation of Chikungunya virus (CHIKV) viral titres obtained by plaque assay using mosquito homogenates at defined time
points. The logarithmic values are also represented.
Aedes aegypti
Time (days) PFU/mL log10 PFU/mL
4 95 1.978
8 1.45 × 102 2.161
12 8 × 103 3.903
16 9.5 × 105 5.978
20 3.5 × 105 5.544
24 1.4 × 105 5.146
28 8.5 × 105 5.929
The CHIKV titre obtained at each time point for each mosquito species by plaque enumeration assay is a mean of triplicate experiments performed with
a single homogenate.
which decreased marginally until the end of the study at 28 days
(8.5 × 105 pfu/mL) (Table 1 and Fig. 4). The titre of infectious
virions obtained from 28 days PI mosquito homogenates was
similar for Ae. aegypti and Ae. albopictus (8.5 × 105 pfu/mL).
No virus was isolated from Cx. quinquefasciatus homogenates
at any time point (Table 1). A Mann–Whitney U-test revealed
that the difference in CHIKV titres observed between Ae.
aegypti and Ae. albopictus mosquitoes was not statistically
significant (U = 16.5; the critical value of U at P < 0.05 is 5;
Z-score = −0.16013; P = 0.87288).
A real-time RT-PCR was performed using whole mosquito
homogenates to detect CHIKV RNA in orally infected
mosquitoes. The positive control (CHIKV infected tissue
culture fluid) in the RT-PCR assays showed a Ct value as low
as 11.77. Uninfected control mosquitoes of all the three species
included as a negative control did not show any amplification
(Fig. 5). RNA isolated at defined time points from infected
Ae. aegypti and Ae. albopictus samples showed positive ampli-
fication, thereby confirming the presence of CHIKV RNA.
No amplification was observed with RNA extracted from Cx.
quinquefasciatus mosquito homogenates, thus ruling out the
presence of CHIKV RNA in these mosquitoes (Fig. 5).
Identification of CHIKV interacting proteins present
on mosquito midgut BBMF by VOPBA followed by MS
To identify the proteins present in mosquito midgut BBMF
that interact with the CHIKV envelope protein, a VOPBA was
carried out. As is evident from Fig. 6, two distinct bands of
approximate molecular mass 32 kDa and 16 kDa were identified
with Ae. albopictus BBMF proteins. Similarly, six protein bands
identified from Ae. aegypti BBMF (∼32, ∼28, ∼26 kDa, ∼16,
∼13 and ∼12 kDa) were found to interact with CHIKV (Fig. 6).
Two common interacting protein bands (∼32 and ∼16 kDa)
identified with both mosquito species were selected for MS
analysis. No CHIKV-interacting protein bands were observed
with Cx. quinquefasciatus midgut BBMF proteins (Fig. 7).
The absence of any interacting protein band(s) in the no-virus
control and presence of a single interacting protein band at
∼37 kDa, corresponding to the presence of GAPDH, confirmed
the specificity of the assay.
The list of proteins generated via LC-tandem MS (MS/MS)
analysis of the tryptic digested protein products obtained on
© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
Understanding chikungunya virus vector competence 7
Aedes albopictus
PFU/mL log10 PFU/mL Culex quinquefasciatus
130 2.113 Not detected
1 × 102 2.000 Not detected
2.1 × 103 3.322 Not detected
1.4 × 104 4.146 Not detected
13.5 × 105 6.130 Not detected
13.5 × 105 6.130 Not detected
8.5 × 105 5.929 Not detected
Fig. 4. Graphical representation of Chikungunya virus (CHIKV) titre
obtained by plaque assay using whole mosquito homogenates (Aedes
aegypti and Aedes albopictus) at defined time points: x-axis: days post
infection; y-axis: logarithmic values of CHIKV titre.
*The CHIKV titre obtained at each time point for each mosquito
species by plaque enumeration assay is a mean of triplicate experiments
performed with a single homogenate.
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry analysis were sorted using an algorithm (Ghosh
et al., 2017). The proteins were arranged initially in the descend-
ing order of ‘scores’, number of unique peptides and peptide
spectrum matches. Subsequently, proteins that met the filter cri-
teria were then inspected manually for the nature of the protein,
molecular weight, region of excision from the gel and likeli-
hood of it being present on the midgut membrane. The final pro-
teins that could serve as putative CHIKV receptors on mosquito
midgut are shown in Tables 2 and 3.
The Ae. albopictus ∼32 kDa band showed a homology with
prohibitin (molecular mass of 29.87 kDa and a homology score
of 3.06) (Table 2). The matching proteins obtained upon mass
spectrometry analysis of the Ae. albopictus ∼16 kDa did not
project a bona fide CHIKV receptor candidate on mosquito
midguts (Table 2). Similarly, LC-MS/MS analysis of Ae. aegypti
∼32 kDa protein band, based on the molecular mass (38.77 kDa)
and homology score (16.21), identified prohibitin as a putative
CHIKV receptor in the midguts (Table 3). Mass spectrometry
analysis of the ∼16 kDa band for Ae. aegypti did not yield any
protein that could be pursued as a putative CHIKV receptor
(Table 3). All of the other proteins were not taken up for
8 A. Ghosh et al.
Fig. 5. Real-time reverse transcriptase-polymerase chain reaction
amplification plots for Chikungunya virus (CHIKV) RNA isolated
from infected mosquitoes at different time points. Amplification plot
for Aedes aegypti samples (A), Aedes albopictus samples (B) and
Culex quinquefasciatus samples (C). Note the amplification for CHIKV
RNA in (A) and (B) but not in (C). [Colour figure can be viewed at
wileyonlinelibrary.com].
subsequent analysis because they did not fulfill the requirements
of the algorithm.
Discussion
The productive infection of an arbovirus in a competent
mosquito vector is a multistep process that includes initiation
of infection in the midgut, spread of infection within the midgut
epithelium, dissemination from the midgut to secondary tissues,
secondary amplification of the virus in various tissues outside
© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
the midgut, infection of salivary glands and sometimes repro-
ductive tissues for vertical transmission to offspring and, finally,
release of the virus into salivary ducts for horizontal trans-
mission to an uninfected vertebrate host (Hardy et al., 1983).
The present study was designed to understand vector compe-
tence of Ae. aegypti and Ae. albopictus mosquitoes for CHIKV
DRDE-06 in terms of infection of the midgut, spread within
the midgut epithelium and dissemination from the midguts to
heads. Although there are reports describing the vector compe-
tence of Aedes sp. mosquitoes for CHIKV (Girod et al., 2011;
Vega-Rúa et al., 2014; Chen et al., 2015; Vega-Rúa et al., 2015)
and the non-permissiveness of Cx. quinquefasciatus mosquitoes
to CHIKV infection (Jupp et al., 1981; Richards et al., 2010;
van den Hurk et al., 2010), the present study was designed to
understand the molecular basis of the selective permissiveness
of Ae. Aegypti and Ae. albopictus and the non-permissiveness of
Cx. quinquefasciatus mosquitoes for CHIKV infection in terms
of identifying the presence of virus interacting proteins on the
mosquito midguts.
The presence of the viral antigen at 2, 6 and 14 days PI
in midgut squashes of orally infected Ae. aegypti and Ae.
albopictus mosquitoes (100%; 5/5) confirmed persistent CHIKV
infection of midguts (Fig. 1). On the other hand, CHIKV
antigen was not observed in midgut squashes prepared from
Cx. quinquefasciatus mosquitoes (0%; 0/5) that had taken an
infectious blood meal at any time-point studied (Fig. 1). The
intensity of fluorescence signifying the presence of CHIKV
infection was similar for Ae. aegypti and Ae. Albopictus, with the
least at 2 days PI and the highest at 14 days PI. It has been widely
reported that infection of mosquito midguts by arboviruses
is persistent in nature (Franz et al., 2015), which was also
observed in the present study. Furthermore, the susceptibility
of Cx. quinquefasciatus mosquitoes to CHIKV is debatable.
Although some studies have reported that CHIKV infected Cx.
quinquefasciatus (Bessaud et al., 2006) and other species of
Culex mosquitoes (Diallo et al., 1999; Chevillon et al., 2008),
other studies have confirmed that this mosquito species is not
a vector for CHIKV (Jupp et al., 1981; Richards et al., 2010;
van den Hurk et al., 2010). This is probably the first report from
India to demonstrate that CHIKV did not infect locally prevalent
Cx. quinquefasciatus mosquitoes under laboratory conditions.
However, previous reports have implicated the mosquitoes from
Culicidae family as vectors that transmit flaviviruses such as
West Nile virus (Mishra et al., 2001) and Japanese encephalitis
virus (Solomon et al., 2000). Our findings reiterate that virus
vector competence is a highly selective phenomenon, with
either the virus selecting a competent arthropod vector for its
transmission or the vector determining which virus it transmits.
Examination of infected Ae. aegypti mosquito midguts showed
that CHIKV antigen was present in the anterior midgut and a
very small region of the posterior midgut 2 days PI. Thereafter,
CHIKV antigen was expressed throughout the infected mosquito
midguts until 14 days PI, albeit predominantly in the ante-
rior portion (Fig. 2). This observation was constant for all
five Ae. aegypti mosquitoes studied at each time point (100%;
5/5). On the other hand, examination of individual CHIKV
infected Ae. albopictus midguts revealed that the viral anti-
gen was distributed throughout the midgut from 2 days PI until
14 days PI (Fig. 2). This observation was again constant for
 Understanding chikungunya virus vector competence 9

Fig. 6. Virus overlay protein binding assay (VOPBA) with Aedes sp. midgut brush border membrane fraction proteins. Midgut membrane proteins
from Aedes albopictus and Aedes aegypti were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose
membrane. Infectious Chikungunya virus (CHIKV) suspension was overlaid on the nitrocellulose membrane. The interacting proteins were detected
by using CHIKV specific polyclonal antibody. (A) Coomassie stained gel containing resolved mosquito membrane proteins. Lane M indicates the
molecular weight marker. Lane Alb contains resolved Ae. albopictus midgut protein fraction and Lane Aeg contains resolved Ae. aegypti midgut protein
fraction. The proteins that were found to interact with CHIKV upon VOPBA are indicated by arrows. (B) VOPBA of Ae. albopictus membrane protein
blots. Two CHIKV interacting protein bands at ∼32 and ∼16 kDa (arrows) were observed with Ae. albopictus midgut protein fraction in lane LI. Lane
NC consists Ae. albopictus midgut proteins that were not incubated with CHIKV suspension, thereby serving as a no-virus control. Lane GAPDH
consists of Ae. albopictus midgut protein fraction that were probed with anti-GAPDH antibody. (C) VOPBA of Ae. aegypti membrane protein blots.
Six Ae. aegypti BBMF protein bands were seen to interact with CHIKV at ∼32, ∼28, ∼26, ∼16 kDa, ∼13 and ∼12 kDa (arrows) in lane L2. Lane NC
consists of Ae. aegypti midgut proteins that were not incubated with CHIKV suspension, thereby serving as a no-virus control. Lane GAPDH consists
of Ae. aegypti midgut protein fraction that were probed with anti-GAPDH antibody. [Colour figure can be viewed at wileyonlinelibrary.com].

Table 2. Results of sequest search peak lists generated from tandem mass spectrometry analysis of Chikungunya virus (CHIKV)-binding proteins
in Aedes albopictus midgut brush border membrane fraction proteins for ∼32 and ∼16 kDa proteins.

Peptide Molecular
Number of Unique Number of spectrum weight
Accession Description Score Coverage Protein Groups Peptides Peptides matches (kDa)

∼32 kDa protein.

AALF014531 60S ribosomal protein 3.23 6.98 1 1 1 1 33.83
AALF003530 Prohibitin 3.07 9.93 1 2 2 2 29.87
AALF013830 Cytochrome C1 2.16 5.9 1 1 1 1 29.4
AALF017733 Uridine phosphorylase 1.6 5.28 1 1 1 1 31.67
∼16 kDa protein.
AALF006450 Cytochrome c oxidase subunit iv 2.32 8.47 1 1 1 2 20.30
AALF024781 60S ribosomal protein L31 2.30 7.26 1 1 1 1 14.57
AALF007120 Ubiquitin 2.21 12.50 1 1 1 1 14.72

all five Ae. albopictus mosquitoes studied at each time point
(100%; 5/5). A similar observation was reported earlier by
Tchankouo-Nguetcheu et al. (2010) where CHIKV (A226V)
was distributed only in the anterior part of the midgut in Ae.
aegypti mosquitoes 7 days PI with near complete infection of
epithelial cells of that region. Variation in the infection patterns
of midgut epithelial cells has been reported previously for dif-
ferent virus–mosquito species combinations. The dengue type-2
virus was not detected in the anterior portion of the midgut of
Ae. albopictus, whereas JEV infected the entire midgut of Cx.
tritaeniorhynchus mosquito species (Mourya & Mishra, 2000;
Zhang et al., 2010). It is also known that the pattern of midgut
infection by CHIKV is different for two strains of Ae. aegypti
mosquitoes (Dong et al., 2016). The basis for differential pattern
of midgut infection for different virus–mosquito combinations
in terms of virus vector competence remains to be understood;
nevertheless, it is accepted that midgut infection is a prerequi-
site for secondary tissue infections. Additionally, in the present

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
10 A. Ghosh et al.

Table 3. Results of sequest search peak lists generated from tandem mass spectrometry analysis of Chikungunya virus (CHIKV)-binding protein in
Aedes aegypti midgut brush border membrane fraction proteins for ∼32 and ∼16 kDa proteins.

Number of Peptide Molecular

Protein Unique Number of spectrum weight
Accession Description Score Coverage Groups Peptides Peptides matches (kDa)

∼32 kDa protein.

AAEL001673 Actin 21.64 26.60 1 8 8 8 41.75
AAEL012282 Prohibitin 16.22 14.12 1 5 5 5 38.776
AAEL005052 Tubulin beta chain 6.39 4.45 1 2 2 2 50.482
∼16 kDa protein.
AAEL003863 Histone H4 30.73 44.66 1 7 7 9 11.404
AAEL015684 Hypothetical protein 5.99 6.01 1 2 2 3 35.887
AAEL007609 Histone H2A 4.77 7.14 1 1 1 2 13.215

midgut barrier, a crucial determinant for establishing infection

Fig. 7. Virus overlay protein binding assay (VOPBA) with Culex
quinquefasciatus midgut brush border membrane fraction proteins.
Midgut membrane proteins from Cx. quinquefasciatus were resolved
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membrane. Infectious Chikungunya virus
(CHIKV) suspension was overlaid on the nitrocellulose membrane. The
interacting proteins were detected by using CHIKV specific polyclonal
antibody. (A) Coomassie stained gel containing resolved mosquito
membrane proteins. Lane M indicates the molecular weight marker.
Lane L1 contains resolved Culex sp. midgut proteins. (B) VOPBA
of Cx. quinquefasciatus membrane protein blots. Lane M represents
the molecular weight marker. Note that no CHIKV interacting Culex
BBMF proteins were detected in lane L2 by VOPBA. Lane NC consists
of Cx. quinquefasciatus midgut proteins that were not incubated with
CHIKV suspension, thereby serving as a no-virus control. Lane GAPDH
consists of Cx. quinquefasciatus midgut protein fraction that were
probed with anti-GAPDH antibody. [Colour figure can be viewed at
wileyonlinelibrary.com].
study, the intensity of fluorescence signifying CHIKV infection,
was the least at 2 and 4 days PI, improved at 6 days PI and high-
est at 10 and 14 days PI, suggesting significant infection in both
Ae. aegypti and Ae. albopictus.
Dissemination efficiency is defined as the proportion of
mosquitoes with virus detected in heads amongst tested ones
(engorged mosquitoes that survived until the day of exam-
ination) (Vega-Rúa et al., 2014). Dissemination efficiency is
therefore a measure of the ability of a virus to overcome the
in an arthropod vector. Both species of Aedes mosquitoes in the
present study showed that CHIKV antigen was present in midgut
squashes of infected Ae. aegypti and Ae. albopictus mosquitoes
(100% positivity; 5/5) at each time point studied from 4 days PI
to 28 days PI (Fig. 3). The viral antigen was absent in midguts of
Cx. quinquefasciatus mosquitoes at all time point studied (0%
positivity; 0/5) (Fig. 3). CHIKV antigen could not be detected
in smears prepared from heads of Aedes sp mosquitoes prior to
8D (Fig. 3). However, from 8 days onwards, all five Aedes sp.
mosquito head specimens tested, at each time point, were posi-
tive for CHIKV antigen by IFA (100% positivity; 5/5).
The intensity of fluorescence denoting CHIKV infection
in Ae. aegypti and Ae. albopictus midguts was observed to
gradually increase from the first time point studied, namely
4 days PI (+), reaching maximum fluorescence (+++) at 12 days
PI. The fluorescence intensity was observed to be constant
for all subsequent time points (Fig. 3). In the case of head
squashes, apple green fluorescence was first detected at 8 days
PI (+), which attained maximum intensity at 16 days PI (+++)
and remained unchanged thereafter (Fig. 3). An earlier study
reported that CHIKV dissemination rates were high for Ae.
aegypti and Ae. albopictus mosquito populations from America
irrespective of the presence of A226V mutation on the E1 gene
of CHIKV (Vega-Rúa et al., 2014). Similarly, in the present
study, CHIKV DRDE-06 (A226) was observed to infect the
midguts of Ae. aegypti and Ae. albopictus mosquitoes found in
South India at 4 days PI and the heads at 8 days PI.
The extrinsic incubation period is defined as the time required
for the virus to be detected in saliva of mosquitoes ready for
transmission after ingestion during the blood meal (Vega-Rúa
et al., 2014). Because a mosquito saliva collection facility
was not accessible, the presence of CHIKV could not be
demonstrated in the saliva of infected mosquitoes.
Furthermore, the number of infectious virions recovered at
each time point (4, 8, 12, 16, 20, 24 and 28 days) from Ae.
aegypti, Ae. albopictus and Cx. quinquefasciatus mosquitoes
was enumerated by plaque assay using whole mosquito
homogenates. A Mann–Whitney U-test demonstrated that there
was no statistically significant difference in the replication kinet-
ics of the virus in the two species of Aedes sp. mosquitoes used
in the present study. However, it was noted that CHIKV attained
a higher titre in Ae. aegypti (16 days) within a lesser time

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376

interval compared with Ae. albopictus mosquitoes (20 days)
(Fig. 4 and Table 1). The real time RT-PCR experiment was
performed to confirm the presence of CHIKV RNA in the
mosquito homogenate preparations (Fig. 5) that were used for
quantifying the infectious virus by plaque enumeration assay.
Overall, the dissemination efficiency and the replication effi-
ciency of CHIKV in Ae. aegypti and Ae. albopictus mosquitoes
in the present study suggested that the virus can infect, repli-
cate in midguts and disseminate to the heads of both species of
Aedes mosquitoes. It would have been interesting to investigate
the length of persistence of the virus beyond 28 days.
The single nucleotide mutation in the CHIKV E1 gene
(A226V) reported during the 2005–06 La Reunion outbreak and
subsequently garnered attention, with studies reporting signifi-
cantly higher infectivity in Ae. albopictus mosquitoes, without
having a considerable effect on CHIKV transmission by Ae.
aegypti (Tsetsarkin et al., 2007). In India, the mutant CHIKV
was isolated and reported during the 2007 epidemic and not
the 2006 outbreak (Arankalle et al., 2007) from the southern
states of Kerala and Karnataka (Santhosh et al., 2009; Kumar
et al., 2012). CHIKV DRDE-06 was obtained during the 2006
epidemic in India and belongs to the Indian Ocean lineage
of East Central and South African genotype (Agarwal et al.,
2014; Kumar et al., 2014). Outbreaks in India and especially
South India are known to be caused by the non-mutant A226
(Gurav et al., 2012; Afreen et al., 2014; Kumar et al., 2014;
Parashar et al., 2015; Naresh et al., 2016), as well as mutant
A226V CHIKV strains (Santhosh et al., 2009; Kumar et al.,
2012; Ray et al., 2012). However, there are no studies from
India on the competence of Ae. albopictus mosquitoes to trans-
mit the non-mutant CHIKV strain compared with Ae. aegypti
mosquitoes. A Taiwan-based study had prevously reported that
CHIKV belonging to the East Central and South African geno-
type, without the A226V mutation, replicated almost similarly
in Ae. aegypti and Ae. albopictus mosquitoes (Chen et al.,
2015). Similarly, efficient transmission of CHIKV (A226) by Ae.
aegypti and Ae. albopictus mosquitoes from different American
countries was observed in a study by Vega-Rúa et al. (2014).
The presence of specific midgut epithelial receptors has
been attributed, at least in part, to the difference in vector
competence for virus transmission between mosquito species
(Mercado-Curiel et al., 2008). It has been documented that most
of the well characterized arbovirus receptors are housekeeping
molecules that are also present ubiquitously in the midgut brush
border membrane of mosquitoes (Paingankar et al., 2010).
CHIKV interacting proteins present amongst the BBMF pro-
teins of Ae. aegypti and Ae. albopictus mosquitoes were iden-
tified by adopting the classical VOPBA technique. Six CHIKV
interacting proteins with Ae. aegypti and two with Ae. albopic-
tus midgut proteins were observed in the present study (Fig. 6).
The two common CHIKV interacting protein bands (∼32 and
16 kDa) from both species of Aedes mosquitoes were processed
for mass spectrometry to identify the proteins. Although anal-
ysis of ∼16 kDa band did not yield a conclusive result, the
∼32 kDa band was found to be prohibitin (Tables 2 and 3).
Prohibitin, a conserved and ubiquitously expressed protein in
eukaryotes has been identified as a CHIKV receptor in human
microglial cells, CHME-5 (Wintachai et al., 2012). Prohibitin
has also been identified as a receptor for dengue type-2 virus in
© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12376
Understanding chikungunya virus vector competence 11
a mosquito cell line (C6/36), as well as on Ae. aegypti mosquito
midguts, by Kuadkitkan et al. (2010). Both Ae. aegypti and
Ae. albopictus mosquitoes are known vectors for CHIKV and
dengue (Kuadkitkan et al., 2010; Wintachai et al., 2012). Fur-
ther experiments are required to determine whether arboviruses
belonging to two different families (Chikungunya: Togaviridae
and Dengue: Flaviviridae) utilize the same receptor protein, pro-
hibitin, on mosquito midguts. Subsequent studies can in turn
help to understand whether these arboviruses use a universal
receptor protein or employ multiple protein/s to infect mosquito
midguts.
In the present study, the non-permissiveness of Cx. quinque-
fasciatus mosquitoes for CHIKV can be partially explained in
terms of absence of CHIKV interacting protein(s) on the midgut
membranes (Fig. 7). Culex quinquefasciatus is reported to be
non-permissive to dengue virus. Liu et al. (2008) demonstrated
the absence of dengue virus (serotype 2) interacting proteins
on Cx. quinquefasciatus midgut membrane using VOPBA. The
findings of the present study therefore support the mesenteron
infection barrier theory (Liu et al., 2008) from pathogenic and
vector competence viewpoints.
The present study unequivocally demonstrates that CHIKV
DRDE-06 infected and replicated similarly in midguts of Ae.
aegypti and Ae. albopictus mosquitoes found in South India. For
the first time, VOPBA experiments performed with mosquito
BBMF preparations reveal that prohibitin could serve as a puta-
tive CHIKV receptor on Aedes mosquito midguts. An absence of
CHIKV specific receptor/s on midguts of Cx. quinquefasciatus
mosquitoes could be one of the reasons why these mosquitoes
are non-permissive to infection. Epidemiologically and ecolog-
ically, it would be interesting to expand this observation with
mosquitoes of same species collected from different parts of
the country. The present study comprised an exercise aiming to
understand different facets of a vast subject, virus vector biology
that has intrigued researchers for many decades. Vector compe-
tence for CHIKV remains less understood even today as a result
of the multiple extrinsic and intrinsic parameters of both the host
species and the virus. Understanding vector competence there-
fore increases the scope with respect to conceptualizing effective
vector control measures that may help to address existing pub-
lic health concerns in a chikungunya endemic country such as
India.
Acknowledgements
This work was funded by a Department of Biotechnology
(DBT), Government of India (GOI) grant entitled ‘Under-
standing the biology of Chikungunya virus infection in per-
missive cell lines and mosquito vectors’ awarded to Desai
(principal investigator) and Tyagi (Co-Principal Investigator)
(102/IFD/SAN/5323/2012–2013 dated March, 15 2013). AG
received a fellowship from the Council of Scientific and Indus-
trial Research (CSIR), Government of India (GOI), Ref no.
19–12/2010 (I) EU-IV, dated June 28, 2011. The data support-
ing the conclusions of this article are included within the article.
AD, VR, AG and BKT designed the study. TM, SB and AG
performed the experiments. AD,VR and AG analysed data.
12 A. Ghosh et al.
AG wrote the manuscript. All authors reviewed and approved
the final manuscript submitted for publication. The authors
declare that they have no conflicts of interest.
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Accepted 25 February 2019