Oral susceptibility of Aedes aegypti (Diptera: Culicidae) from

Senegal for dengue serotypes 1 and 3 viruses

Alioune Gaye
1,2
, Oumar Faye
3
, Cheikh T. Diagne
1,2
, Ousmane Faye
3
, Diawo Diallo
1
, Scott C. Weaver
4
,
Amadou A. Sall
4
and Mawlouth Diallo
1
1 Unite dentomologie Medicale, Institut Pasteur de Dakar, Dakar, Senegal
2 Universite Cheikh Anta Diop de Dakar, Dakar, Senegal
3 Unite des Arbovirus et virus de Fievres Hemorragiques, Institut Pasteur de Dakar, Dakar, Senegal
4 Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA
Abstract objective To investigate the potential for domestic and wild populations of Aedes aegypti from
Dakar and Kedougou to develop a disseminated infection after exposure to DENV-3 and DENV-1.
methods We have exposed sylvatic and urban population of Ae. aegypti from Senegal to bloomeals
containing dengue serotype 1 and 3. At different incubation period, individual mosquito legs/wings
and bodies were tested for virus presence using real time RTPCR to estimate the infection and
dissemination rates.
results The data indicated low susceptibility to DENV-3 (infection: 2.415.2%, and dissemination
rates: 08.3%) and higher susceptibility to DENV-1 (infection and dissemination rates up to 50%).
conclusion Aedes aegypti from Senegal seem able to develop a disseminated infection of DENV-1
and DENV-3. Further studies are needed to test their ability to transmit the two serotypes.
keywords Aedes aegypti, Dakar, Kedougou, Senegal, oral susceptibility, dengue serotypes 1 and 3
Introduction
Dengue virus (DENV) remains a major public health
problem in tropical regions with an estimate of 390 mil-
lions of infections each year, comprised of 96 million
apparent and 294 million non-apparent infections (Bhatt
et al. 2013). Dengue is caused by four genetically differ-
ent (DENV 14) serotypes of viruses (genus Flavivirus,
family Flaviviridae). DENV is primarily transmitted by
the mosquito Aedes aegypti and secondarily by Aedes
albopictus. The time between entrance of the virus in the
vector and the moment when it can be transmitted is
known as extrinsic incubation period (EIP) and varies
between 8 and 12 days for all serotypes (Gubler et al.
1979; Gubler 1998).
Recently, major epidemiologic changes have been
recorded in Africa with DENV-3 outbreaks in C^ ote
dIvoire in 2008, in Senegal and in Cape Verde in 2009
(Amarasinghe et al. 2011). Before 2007, only DENV-1,
DENV-2 and DENV-4 were known to circulate in Sene-
gal. Sylvatic DENV-2 amplications have been regularly
observed for decades in south-eastern Senegal (Diallo
et al. 2003). Only three DENV-4 human cases were
reported in Senegal from Europeans (Saluzzo et al.
1986); there are no data on the mosquito vectors
involved. Serological data indicate outbreaks of DENV-1
in several cities in Africa (Amarasinghe et al. 2011). In
contrast, DENV-3 was detected more recently in 2007 in
Spain in an immigrant returning from Senegal (Amarasin-
ghe et al. 2011) and in October 2009 during an epidemic
in Dakar and Mbour (Faye et al. 2014).
There are many reports globally about vector compe-
tence of Aedes aegypti and Aedes albo-pictus for dengue
1 and 3 viruses (Table 1), but none on the susceptibility
of Senegalese mosquitoes for DENV 1, 3 and 4, and
only a few on DENV-2. Therefore, in this study, we
assessed the oral susceptibility of domestic and wild pop-
ulations of Ae. aegypti from Senegal for DENV-3 and
DENV-1.
Materials and methods
Two populations of Ae. aegypti were used: a purely syl-
vatic and zoophilic population collected in the forest gal-
lery of Kedougou (1233
0
00 N, 1211
0
00 W) breeding
in tree holes and a domestic and anthropophilic popula-
tion collected in the urban environment of Dakar
(1443
0
29 N, 1728
0
24 W) breeding in articial con-
tainers. Morphologically, the lack and presence of
pale scales on the rst abdominal tergite differentiate,
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Tropical Medicine and International Health doi:10.1111/tmi.12373
volume 00 no 00
respectively, the population from Kedougou related to
Ae. aegypti formosus and Dakar compatible with Ae. ae-
gypti aegypti.
DENV1_IbH28328 strains isolated from human sera
from Ibadan (Nigeria) in 1964 and DENV3 H87 strains
isolated from human sera from Hawaii in 1957 were
used. The virus stocks were prepared using brains of
newborn mice showing signs of illness after intracerebral
inoculation of 0.02 ml of DENV1 or DENV3.
The F1 generation, 4- to 5-day-old female mosquitoes
starved for 2448 h, was exposed to articial infectious
blood meal for 30 min as previously described (Diallo
et al. 2008). Fully engorged mosquitoes (n = 276) were
selected and incubated at 27 1C, 80 5% RH, and
Table 1 Chronological worldwide reports about vector competence of Aedes aegypti and Aedes albopictus for dengue 1 and 3 viruses
Serotypes
Origin and history of the virus strains used Origin of mosquitoes used
Test
done References Country/host/year Passage history Species Geographic origin
DENV1 Unknown Unknown Ae. albopictus Vietnam, Madagascar,
Malaysia, India, Taiwan,
Thailand, Hawaii,
Mauritius, Indonesia,
Philippine
ST Gubler &
Rosen (1976)
Unknown Unknown Ae. aegypti South Pacic, Indonesia,
Malaysia, Singapore,
Philippine, Thailand,
Burma, Kenya, Burkina
ST Gubler et al. (1979)
Fiji/human/1975 1 C6/36 Ae. albopictus Malaysia, Japan, Texas,
Tennessee, Louisiana
OT Boromisa et al. (1987)
Fiji/human/1975 1 C6/36 Ae. aegypti Texas (USA) OT Boromisa et al. (1987)
Puerto Rico/human/1985 1 Mosquito Ae. albopictus Houston (USA) OT Mitchell et al. (1987)
Puerto Rico/human/1985 1 Mosquito Ae. aegypti Rexville (USA) OT Mitchell et al. (1987)
Fiji/human/1975 1 C6/36, 2 Toxo Ae. albopictus Hawaii VT Shroyer (1990)
Puerto Rico/human/1985 1 Toxo Ae. albopictus Brazil VT Mitchell &
Miller (1990)
Taiwan/human/1987 1 Toxo,
1LLCMK2
3 Vero, 1C6/36
Ae. albopictus
Ae. aegypti
Taiwan OT Chen et al. (1993)
Durban/human/1985 3 Mice, 4 mosq Ae. aegypti South Africa ST Jupp and Kemp (1993)
Australia/human/1990 1 C6/36 Ae. aegypti Australia OT Watson & Kay (1999)
Unknown Unknown Ae. albopictus China ST Shu et al. (2004)
Florida/human/2010 1 AGM, 2 Vero Ae. aegypti
Ae. albopictus
Florida (USA) OT Richards et al. (2012)
Florida/human/2010 1 AGM, 2 Vero Ae. aegypti
Ae. albopictus
Florida (USA) OT Buckner et al. (2013)
DENV3 Unknown Unknown Ae. aegypi South Pacic, Indonesia,
Malaysia, Singapore,
Thailand, Burma,
Philippines, Kenya,
Burkina
OT Gubler et al. (1979)
Mozambique/
human/1985
1 Mosq, 1 C3/36 Ae. albopictus Houston (USA) OT Mitchell et al. (1987)
Mozambique/
human/1985
1 Mosq, 1 C3/36 Ae. aegypi Rexville (USA) OT Mitchell et al. (1987)
Unknown Unknown Ae. aegypti India VT Joshi et al. (1996)
Philippines/human/1956 1 Mice, 1 C6/36 Ae. aegypti Australia ST Watson & Kay (1999)
Thailand/human/1963 21 Mouse Ae. aegypti India VT Joshi et al. (2002)
Unknown Unknown Ae. albopictus China ST Shu et al. (2004)
Cape Verde/
Human/2009
1 C6/36 Ae. aegypti Cape Verde OT Vazeille et al. (2013)
Toxo, toxorhynchites; Mosq, mosquito; OT, oral transmission; VT, vertical transmission; ST, susceptibility test.
2 2014 John Wiley & Sons Ltd
Tropical Medicine and International Health volume 00 no 00
A. Gaye et al. Susceptibility of Ae. aegypti to dengue
fed with 10% glucose. Samples of mosquitoes were col-
lected 7, 15 and 20 days post-infection (dpi), cold
anesthetised and dissected (n = 252 i.e. a mortality rate
of 8.7%). Bodies (head-abdomen) and leg wings were
separately triturated in 500 ll L15 medium and tested
by real-time RT-PCR for DENV detection using the
Qiagen One-step kit (Qiagen Inc., Santa Clarita, CA).
The reaction mixture consisted of 5 ll RNA, 10 ll of
buffer (2 X QuantiTect Probe), 6.8 ll of RNase free
water, 1.25 ll each primer, forward (5
0
ATTA-
GAGAGCAGATCTCTG 3
0
) and reverse (5
0
TGA-
CACGCGGTTTC 3
0
), 0.5 ll of probe (5
0
TCAATATGCTGAAACGCG 3
0
), and 0.2 ll of enzymes
to a total volume of 25 ll. The RT-PCR was performed
by ABI Prism 7500 SDS (Applied Biosystems, Foster City,
USA). The cycling conditions were RT step at 50.0 C
for 10 min, at 95.0 C for 15 min, and 40 cycles of 15 s
at 95.0 C and 1 min at 60 C. Infection (number of
positive bodies/total number of bodies tested) and dissem-
ination (number of infected legs wings / number of
DENV-positive bodies) rates were calculated. Fishers
tests were performed using Epi-Info version 6.04 (CDC,
Atlanta, GA, USA) for comparison of infection and
dissemination.
Results
Infection and dissemination rates obtained for DEN 1
and 3 are summarised in Table 2. The small sample size
of Aedes aegypti from Kedougou tested in some dpi may
have impacted the results observed for this population.
This impact could be minimised by acceptable number of
specimens tested at 15 dpi.
Regarding DENV-3, the infection rates of the domestic
Ae. aegypti population ranged from 2.4 to 15.2% with a
signicant difference between the rates obtained at 15
and 20 dpi (P = 0.03). While infected early (7 dpi), they
disseminated DENV by 15 dpi with a rate of 8.3%. The
forest population exhibited only non-disseminated infec-
tions at 15 dpi.
With DENV-1, the Ae. aegypti populations from
Dakar exhibited infection rates ranging from 0 to 43.8%
and the dissemination from 7.1% to 50%. These rates
were statistically similar (P = 0.51 and P = 0.11, respec-
tively). For the Ae. aegypti population from Kedougou,
infection rates with DENV-1 varied from 30% to 50%,
but disseminated infections were only observed at 20 dpi
at a frequency of 33.3%. No signicant differences were
observed between mosquito populations.
The comparison of infection and dissemination rates,
in the two Ae. aegypti populations for each DENV sero-
type and at each incubation period, did not reveal signi-
cant differences. The two Ae. aegypti populations showed
higher infection and dissemination rates with DENV-1
compared with DENV-3. The differences were signicant
at 15 and 20 dpi for Ae. aegypti from Dakar
(P < 0.009).
Discussion
Our ndings revealed relatively low DENV-3 infection
and dissemination rates in the two Senegalese populations
of Ae. aegypti, compared with mosquitoes from Austra-
lia, Asia and Cape Verde (Gubler et al. 1979; Vazeille
et al. 2013). The oral virus titres used do not explain this
difference because similar infection rates (012.5%) were
obtained with Ae. aegypti from Burkina Faso and Kenya
exposed to a virus titre 10
7.3
MID
50
/ml. Our data are
comparable with those obtained with several populations
of Ae. aegypti from dengue-endemic locations such as
Table 2 Infection and dissemination rates of Aedes Aegypti populations from Dakar and Kedougou orally exposed to DENV-1 and
DENV-3
Species Virus strain
Blood meal titre
(MID50/ml)
Infection and dissemination according to each extrinsic incubation period
or day post-infection (dpi)
Infection rate (%) Dissemination rate (%)
7 15 20 7 15 20
Aedes aegypti
(DKR)
DENV1_IbH28328 5.10
3.3
0/9 14/32 (43.7) 4/13 (30.8) NT 1/14 (7.14) 2/4 (50)
DENV3 H87 5.10
4.4
4/40 (10) 12/79 (15.2) 1/41 (2.4) 0/4 1/12 (8.3) 0/1
Aedes aegypti
(KDG)
DENV1_IbH28328 5.10
4.3
2/5 (40) 3/10 (30) 3/6 (50) 0/2 0/3 1/3 (33.3)
DENV3 H87 5.10
4.2
0/5 1/12 (8.3) NT NT 0/1 NT
(): percentage of infection [number of positive bodies/total number of bodies incubated after engorgement] and dissemination [number
of infected legs wings/number of DENV-positive bodies] rates; MID, mice infection dose; KDG, Kedougou; DKR, Dakar; NT, not
tested.
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Tropical Medicine and International Health volume 00 no 00
A. Gaye et al. Susceptibility of Ae. aegypti to dengue
Manila and Singapore, which developed 16.918.7%
infection rates with a virus titre 10
6.7
MID
50
/ml (Gubler
et al. 1979), or Thai mosquitoes, where an infectious
blood meal 10
7
MID
50
/ml generated 0.919.7% infec-
tion (Thongrungkiat et al. 2003). In the absence of an
accurate measure of threshold required to infect mosquito
with dengue viruses, other factors, such as the geographic
origin of the mosquito strain, may explain these
differences.
Dengue virus-1 seems to be more infectious for both
Senegalese populations tested. Our ndings revealed sus-
ceptibilities comparable with those described for Ae. ae-
gypti from dengue-endemic locations (Gubler et al. 1979;
Thongrungkiat et al. 2003; Vazeille et al. 2013). How-
ever, their dissemination rates are low compared with
populations from South Africa (Jupp & Kemp 1993) and
Taiwan (Chen et al. 1993). The drops in infection rates
after 15 dpi were probably due to virus clearance by the
mosquito immune system (Sanchez-Vargas et al. 2009).
This preliminary study indicates the ability of Ae. ae-
gypti from Senegal to develop disseminated infections of
DENV-1 and DENV-3. Further studies are necessary to
test its ability to transmit the two serotypes and to deter-
mine the parameters controlling this transmission. The
decrease of infection rates after a long incubation period
and the delayed dissemination observed in some cases, as
well as the important role of sylvatic mosquitoes in
DENV-2 transmission in Africa, need further investigation.
Acknowledgements
We are grateful to Amadou Thiaw, Abdou Karim Bodian
of the Unite dEntomologie Medicale at Institut Pasteur
de Dakar as well as our eld and staff in Kedougou for
their technical assistance. This research was supported by
an NIH Grant. RO1AI069145.
References
Amarasinghe A, Kuritsky JN, Letson GW & Margolis HS
(2011) Dengue virus infection in Africa. Emerging Infectious
Diseases 17, 13491354.
Bhatt S, Gething PW, Brady OJ et al. (2013) The global distribu-
tion and burden of dengue. Nature 496, 504507.
Boromisa RD, Rai KS & Grimstad PR (1987) Variation in the
vector competence of geographic strains of Aedes albopictus
for dengue 1 virus. Journal of the American Mosquito Control
Association 3, 378386.
Buckner EA, Alto BW & Lounibos LP (2013) Vertical transmis-
sion of Key West dengue-1 virus by Aedes aegypti and Aedes
albopictus (Diptera: Culicidae) mosquitoes from Florida. Jour-
nal of Medical Entomology 50, 12911297.
Chen WJ, Wei HL, Hsu EL & Chen ER (1993) Vector compe-
tence of Aedes albopictus and Ae. Aegypti (Diptera: Culicidae)
to dengue 1 virus on Tawan: development of the virus in
orally and parenterally infected mosquitoes. Journal of Medi-
cal Entomology 30, 524530.
Diallo M, Ba Y, Sall AA et al. (2003) Amplication of the syl-
vatic cycle of dengue virus type 2, Senegal, 19992000: ento-
mologic ndings and epidemiologic considerations. Emerging
Infectious Diseases 9, 362367.
Diallo M, Ba Y, Faye O et al. (2008) Vector competence of
Aedes aegypti populations from Senegal for sylvatic and epi-
demic dengue 2 virus isolated in West Africa. Transactions of
the Royal Society of Tropical Medicine and Hygiene 102,
493498.
Faye O, Ba Y, Faye O et al. (2014) Major urban dengue 3 epi-
demic in Senegal in 2009. Emerging Infectious Diseases 20,
456459.
Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clini-
cal Microbiology Reviews 11, 480496.
Gubler DJ & Rosen L (1976) Variation among geographic
strains of Aedes albopictus in susceptibility to infection with
dengue viruses. American Journal of Tropical Medicine and
Hygiene 25, 318325.
Gubler DJ, Nalim S, Tan R, Saipan H & Sulianti Saroso J
(1979) Variation in susceptibility to oral infection with dengue
viruses among geographic strains of Aedes aegypti. American
Journal of Tropical Medicine and Hygiene 28, 10451052.
Joshi V, Singhi M & Chaudhary RC (1996) Transovarial trans-
mission of dengue 3 virus by Aedes aegypti. Transactions of
the Royal Society of Tropical Medicine and Hygiene 90, 643
644.
Joshi V, Mourya DT & Sharma RC (2002) Persistence of den-
gue-3 virus through transovarial transmission passage in suc-
cessive generations of Aedes aegypti mosquitoes. American
Journal of Tropical Medicine and Hygiene 67, 158161.
Jupp PG & Kemp A (1993) The potential for dengue in South
Africa: vector competence tests with dengue 1 and 2 viruses
and 6 mosquito species. Transactions of the Royal Society of
Tropical Medicine and Hygiene 87, 639643.
Mitchell CJ & Miller BR (1990) Vertical transmission of dengue
viruses by strains of Aedes albopictus recently introduced into
Brazil. Journal of the American Mosquito Control Association
6, 251253.
Mitchell CJ, Miller BR & Gubler DJ (1987) Vector competence
of Aedes albopictus from Houston, Texas, for dengue sero-
types 1 to 4, yellow fever and Ross River viruses. Journal of
the American Mosquito Control Association 3, 460465.
Richards SL, Anderson SL & Alto BW (2012) Vector compe-
tence of Aedes aegypti and Aedes albopictus (Diptera: Culici-
dae) for dengue virus in the Florida Keys. Journal of Medical
Entomology 49, 942946.
Saluzzo JF, Cornet M, Adam C, Eyraud M & Digoutte JP
(1986) Dengue 2 au Senegal oriental: enqu^ete serologique dans
les populations simiennes et humaines. 19741985. Bulletin
de la Societe de Pathologie Exotique et de ses Filiales 79,
313322.
4 2014 John Wiley & Sons Ltd
Tropical Medicine and International Health volume 00 no 00
A. Gaye et al. Susceptibility of Ae. aegypti to dengue
Sanchez-Vargas I, Scott JC, Poole-Smith BK et al. (2009) Dengue
virus type 2 infections of Aedes aegypti are modulated by the
mosquitos RNA interference pathway. PLoS Pathogens 5,
e1000299.
Shroyer DA (1990) Vertical maintenance of dengue-1 virus in
sequential generations of Aedes albopictus. Journal of the
American Mosquito Control Association 6, 312314.
Shu LP, Zuo L, Zhao X, Chen AY & Wei LH (2004) Suscepti-
bility of 15 collections of Aedes albopictus from Guizhou to
dengue virus oral infection. Zhonghua Shi Yan He Lin Chuang
Bing Du Xue Za Zhi 18, 234237.
Thongrungkiat S, lirakanjanakit N, Apiwathnasom C et al.
(2003) Comparative susceptibility to oral infection with
dengue viruses among local strains of Aedes aegypti (Diptera:
Culicidae) collected at different seasons of the year 2003. Jour-
nal of Vector Ecology: Journal of the Society for Vector Ecol-
ogy 28, 166170.
Vazeille M, Yebakima A, Lourenc o-de-Oliveira R et al. (2013)
Oral receptivity of Aedes aegypti from Cape Verde for yellow
fever, dengue, and chikungunya viruses. Vector Borne and
Zoonotic Diseases 13, 3740.
Watson TM & Kay BH (1999) Vector competence of Aedes not-
oscriptus (Diptera: Culicidae) for Barmah Forest virus and of
this species and Aedes aegypti (Diptera: Culicidae) for dengue
1-4 viruses in Queensland, Australia. Journal of Medical
Entomolgy 36, 508514.
Corresponding Author Mawlouth Diallo, Unite dEntomologie Medicale, Institut Pasteur de Dakar, BP 220 Dakar, Senegal.
Tel.: +221.338399228; Fax +221.338399210; E-mail: diallo@pasteur.sn
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