Vol. 40, no.

1 Journal of Vector Ecology 71

Dengue virus detection in Aedes aegypti larvae from southeastern Brazil

Samyra Giarola Cecílio1*, Willer Ferreira Silva Júnior1, Antônio Helvécio Tótola2,
Cíntia Lopes de Brito Magalhães3, Jaqueline Maria Siqueira Ferreira4, and José Carlos de Magalhães1
¹Laboratório de Microbiologia Geral e Enzimologia, samyracecilio@gmail.com
2
Laboratório de Bioquímica e Imunologia Celular e Molecular, Departamento de Química, Biotecnologia e Engenharia de Bioprocessos
(DQBIO/Campus Alto Paraopeba/UFSJ), Ouro Branco, MG, Brazil
3
Laboratório de Biologia e Tecnologia de Micro-organismos, Departamento de Ciências Biológicas, (DCB/UFOP), Ouro Preto, MG, Brazil
4
Laboratório de Microbiologia (Campus Centro Oeste Dona Lindu/UFSJ), Divinópolis, MG, Brazil

Received 11 August 2014; Accepted 3 December 2015

ABSTRACT: The transmission of dengue, the most important arthropod-borne viral disease in Brazil, has been intensified over the past
decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors
in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between
September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified
according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs
was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index
(EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was
low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four
Ae. aegypti pools. Journal of Vector Ecology 40 (1): 71-74. 2015.

Keyword Index: Aedes, dengue virus, ovitraps, transovarial transmission, oviposition, mosquitoes.

INTRODUCTION has epidemiological importance in disease maintenance by the

Dengue virus (DENV) has been the most important arboviral
disease in the world, responsible for significant morbidity and
mortality, especially in tropical countries (Bhattacharya et al.
2013, Khan et al. 2013, Restrepo et al. 2014). Dengue is caused by
the four antigenically distinct serotypes of the virus (DENV 1-4),
which belong to the genus Flavivirus, family Flaviviridae, and is
transmitted by infected Aedes mosquitoes (Añez and Rios 2013,
Restrepo et al. 2014). Aedes aegypti is the primary vector, followed
by Aedes albopictus, which also transmit the viruses causing West
Nile, yellow fever, and chikungunya fevers (Regis et al. 2008,
Primavesi 2013, Restrepo et al. 2014). The predilection of females
for human blood enhance disease transmission among humans
(Carrington and Simmons 2014). Illness with any of the four
serotypes results in wide range of symptoms, from a milder dengue
fever (DF) to the serious and often fatal dengue hemorrhagic fever
(DHF) and hemorrhagic shock syndrome (DSS) (Gubler 1998).
The disease brings considerable health, social, and economic
problems to endemic areas. With the ongoing search for an
efficient vaccine and an antiviral drug, prevention is still the
best way to control the disease, which is possible through vector
control in several forms (Bäck and Lundkvist 2013, Resende et
al. 2012, Resende et al. 2013). Various trap models to capture and
measure the density of the vector have been designed, and their
use in many countries has shown them to be a better strategy than
classical surveillance methods for monitoring (Regis et al. 2008,
Regis et al. 2013).
Ovitraps, or mosquito egg traps, exhibit a great sensitivity
even at low vector densities (Santos et al. 2003). Moreover,
they can detect transovarial transmission of the virus, which
possibility of establishing new outbreaks in non-epidemic periods
(Vélez et al. 1998, Kow et al. 2001).
Thus, the present study was designed to assess the circulation
of Ae. aegypti and Ae. albopictus vectors with the Ovitrap Positivity
Index (OPI) and Eggs Density Index (EDI), and detect dengue
virus transovarial transmission in Ouro Branco and Ouro Preto,
Brazil.
MATERIALS AND METHODS
Study area and experimental design
Ouro Branco and Ouro Preto are located in southeastern
Brazil in Minas Gerais state, with estimated populations of 36,000
and 70,000, respectively. Sixteen public schools from each district,
in partnership with local health surveillance departments, were
chosen to compose the study, totaling 32 collection points. The
ovitrap installation was conducted from September, 2011 to
July, 2012 and from November, 2012 until May, 2013, totaling 17
months of collection.
The traps consisted of a black plastic container (15 x 10 cm).
In each one, three wooden paddles were fixed with clips for egg
deposition. In order to improve the attractiveness, the ovitraps
were filled with 500 ml of 10% grass infusion (Panicum maximum,
Jacq), prepared according to Reiter et al. (1991). One trap was
installed monthly per school, remaining at the institution for seven
days to exclude the possibility of becoming a source of adults. Eggs
were counted, hatched, and larvae were identified to species (Ae.
aegypti or Ae. albopictus) at the 4th instar. They were separated into
pools up to 40 larvae according to the month and site of collection
and stored at -80° C. The entomological indicators calculated were
72 Journal of Vector Ecology June 2015
OPI (positive traps/traps installed x 100) and EDI (eggs collected/
traps installed).
RNA extraction and RT-PCR
Pools were macerated with the UNIQUE® ultrasonic tissue
macerator. Total RNA was extracted using the RNeasy QIAGEN
Kit® according to the manufacturer’s protocol. As a positive
control, RNA extracted from the supernatant taken from DENV2
infected C6/36 cells was used.
The presence of viral RNA was detected using reverse
transcription-polymerase chain reaction (RT-PCR). For the
synthesis of complementary DNA (cDNA), the reaction mixture
contained 5.0 µl of each extracted RNA, 1.0 μl of the reverse
primer (10 pmol) (Chien et al. 2006), 0.5 μl of RiboLock RNase
Inhibitor (40 U/μl) Fermentas® and 7.25 μl of nuclease-free water
Fermentas®. It was incubated for 5 min at 70° C and 5 min at 4°
C. Subsequently, it was added to a mixture of 1.5 μl of dNTPs (10
mM) Fermentas®, 0.75 μl of M-MLV reverse transcriptase (200
U/μl) - Fermentas® and 4 μl of M-MLV Reaction Buffer  5X –
Fermentas®. The incubation was carried out for 60 min at 42° C.
The reaction was performed in Life-BIOER Express®.
The reaction was carried out on StepOne™, Applied
Biosystems®. The reaction mixture contained 2 μl of cDNA, 3 μl of
each primer (2 pmols) (Chien et al. 2006), 10 μl of Power SYBR-
PCR Master Mix-Applied Biosystems®, and 2 μl of nuclease-
free water Fermentas®, totaling 20 μl per sample. The reaction
followed the steps of an initial denaturation at 95° C for 10 min,
40 amplification cycles of 15 s at 95° C, and 1 min at 60° C. cDNA
from infected C6/36 cells was used as a positive control and sterile
water as a negative control.
PCR products were analyzed by electrophoresis in 1.5%
agarose gels to confirm the size of amplicons. Ten μl were
electrophoresed at 80 V for 1 h in Tris-acetate-EDTA (TAE)
pH 8.3. GeneRuler 50 bp  DNA Ladder Fermentas® was used as
a molecular weight marker. The gel was stained with 0.1 μl/ml
ethidium bromide solution and photographed under ultraviolet
light.
RESULTS
Ovitrap installation
During the 17 months of trap operation, 1,945 eggs were
collected: 812 in Ouro Branco and 1,133 in Ouro Preto. Values of
OPI ranged from 0 to 28.1%, with the highest values in January to
April, 2013. EDI values ranged from 0 to 59.9%, with the highest
values found in December, 2012 and January, 2013. Both cities
presented larger numbers of Ae. aegypti. Of the 1,113 hatched
eggs (57.2%), 945 (84.9%) were identified as Ae. aegypti and 168
(15.1%) as Ae. albopictus. The larvae were distributed in 54 pools
according to the point of collection/date and species.
Real-time PCR and agarose gel electrophoresis
Of the 54 pools, four of Ae. aegypti showed amplification after
the 24th- 30th cycle in real time PCR. Samples were electrophoresed
to confirm the size of amplicons. Primers designed by Chien et
al. (2006) amplify a segment of 146 bp. These bands could be
visualized in agarose gels, corresponding to DENV, confirming
transovarial transmission. Of the four positive pools, three were
Figure 1. Agarose gel electrophoresis of the amplified products by
real-time PCR. Lane 1: standard molecular weight of 50 bp; lane
2: dengue virus 2 control; lanes 3-9: cDNA samples from eggs
collected at different schools. Identification of 146 bp fragments
in lanes 2, 3, 4, 5, 6, and 9 positive for dengue virus. There are also
fragments of ± 50 bp, relative to dimers of primers.
from O. Branco and one was from O. Preto schools. The pools
were from eggs collected in January to April, 2013. As shown
in Figure 1, the DNA samples analyzed by electrophoresis on
agarose gel stained with ethidium bromide, with bands of 146 bp
corresponding to DENV. Some lightly stained nonspecific bands
can also be observed which are fragments of ± 50 bp, relative to
dimers of primers.
DISCUSSION
The present study aimed to monitor Aedes by ovitrap
installation. Devices that detect an increase in population of
these vectors are valuable for health surveillance departments to
better plan their control actions, find areas with higher incidence,
and prevent disease outbreaks. Due to vertical transmission and
resistance of eggs to long periods of dessication, it is important to
focus the prevention of dengue on Aedes eggs.
Oviposition traps have been used in several studies to
monitor vector populations (Almeida et al. 2006, Fantinatti et al.
2007, Costa et al. 2008, Miyazaki et al. 2009, Carvalho-Leandro et
al. 2010, Resende et al. 2013). Braga et al. (2000) and Cardoso et al.
(1997) found higher sensitivity from oviposition traps than from
larval surveys to detect Ae. aegypti. This methodology was more
efficient than adult traps (Gama et al. 2007, Honorio et al. 2009).
In the present study, although egg collection had been low
(higher OPI 28.13 – April, 2013 and EDI 59.9 – January, 2013)
when compared with other studies with ovitraps (Resende et al.
2013, Zeidler et al. 2008, Nunes et al. 2011), data are consistent
with historical cases in both cities, neither of which prepare
epidemic registers and are cold and mountainous. Moreover,
there were peaks in the numbers of eggs collected in the warmer
months of the year, while the other months had much lower or
even null rates. This could allow the detection of fluctuations in
vector density, suggesting that warmer months have an increased
circulation of these arthropods, with a higher risk of disease
transmission.
Vector-borne infectious diseases are generally seasonal.
Indeed, in Brazil, dengue has a seasonal pattern, with a higher
incidence of cases in the first five months of the year that are
Vol. 40, no. 1 Journal of Vector Ecology 73
warmer and wetter (Reiter 2001, Zell 2004). According to Morin et
al. 2013, temperature is an essential element for dengue epidemics.
This variable affects the proliferation of both the vector and virus.
With the increase of daily minimum and maximum temperatures,
there is a faster development of larval populations and the rate
of pathogen replication within the insect (shorter extrinsic
incubation period) (Russell 1998, Gubler et al. 2001, Reiter 2001).
Transovarial transmission of dengue virus occurs both in
the laboratory and in the field, playing an important role in the
dissemination of the disease. Vertical transmission in Ae. aegypti
and Ae. albopictus larvae was detected in studies by Cecilio et al.
(2009) and Pessanha et al. (2011). According to Joshi et al. (2002),
vertical transmission in nature does not occur in more than
20% of the progeny. In this work, using real time PCR, DENV
amplification could be detected in four of the 54 pools of Ae. aegypti
larvae hatched in the laboratory. Considering this small rate of
vertical transmission occurrence, the positivity of pools (7.41%)
was high and the results obtained are of great importance. Results
were passed on to local health surveillance departments that have
committed to plan a greater intervention in these locations.
REFERENCES CITED
Almeida, P.S., A.D. Ferreira, V.L Pereira, M.G Fernandes, and W.D.
Fernandes. 2006. Distribuição espacial de Aedes albopictus na
região sul do Estado de Mato Grosso do Sul. Rev. Saúde Públ.
40: 1094-1100.
Añez, G. and M. Rios. 2013. Dengue in the United States of
America: A worsening scenario? Biomed. Res. Int. 2013: 1-13.
Bӓck, A.T. and A. Lundkvist. 2013. Dengue viruses: an overview.
Infect. Ecol. Epidemiol. 3: 1-21.
Bhattacharya, M.K., S. Maitra, A. Ganguly, A. Bhattacharya, and
A. Sinha. 2013. Dengue: A growing menace - a snapshot of
recent facts, figures and remedies. Int. J. Biomed. Sci. 9: 61-67.
Braga, I.A., A.C. Gomes, M. Nelson, R.C.B. Melo, D.P. Bergamaschi,
and J.M.P. Souza. 2000. Comparação entre pesquisa larvária e
armadilha de oviposição para detecção de Aedes aegypti. Rev.
Soc. Bras. Med. Trop. 33: 347-353.
Cardoso Jr., R.P., S.A.S. Scandar, N.V. Mello, S. Ernandes, M.V.
Botti, and E.M.M. Nascimento. 1997. Detecção de Aedes
aegypti e Aedes albopictus na zona urbana do município de
Catanduva-SP, após controle de epidemia de dengue. Rev.
Soc. Bras. Med. Trop. 30: 37-40.
Carrington, L.B. and C.P. Simmons. 2014. Human to mosquito
transmission of dengue viruses. Front. Immunol. 5: 1-8.
Carvalho-Leandro, D., A.L.M. Ribeiro, J.S.V. Rodrigues, C.M.R.
Albuquerque, A.M. Acel, F.A. Leal-Santos, D.P. Leite Jr., and
R.D. Miyazaki. 2010. Temporal distribution of Aedes aegypti
Linnaeus (Diptera, Culicidae), in a hospital in Cuiabá, State of
Mato Grosso, Brazil. Rev. Bras. Entomol. 54: 701–706.
Cecílio, A.B.,  E.S. Campanelli, K.P.  Souza, L.B.  Figueiredo,
and M.C. Resende. 2009. Natural vertical transmission by
Stegomyia albopicta as dengue vector in Brazil. Braz. J. Biol.
69: 123-127.
Chien, L.J., T.L. Liao, P.Y. Shu, J.H. Huang, D.J. Gubler, and G.J.
Chang. 2006. Development of Real-time reverse transcriptase
PCR assays to detect and serotype dengue viruses. J. Clin.
Microbiol. 44: 1295–1304.
Costa, F.S., J.J. Silva, C.M. Souza, and J. Mendes. 2008. Dinâmica
populacional de Aedes aegypti (L) em área urbana de alta
incidência de dengue. Rev. Soc. Bras. Med. Trop. 41: 309-312.
Fantinatti, E.C.S., J.E.L. Duque, A.M. Silva, and M.A. Navarro-
Silva. 2007. Abundância e Agregação de Ovos de Aedes
aegypti L. e Aedes albopictus (Skuse) (Diptera: Culicidae) no
Norte e Noroeste do Paraná. Neotrop. Entomol. 36: 960-965.
Gama, R.A., E.M. Silva, I. M. Silva, M.C. Resende, and A.E. Eiras.
2007. Evaluation of the Sticky MosquiTRAPTM for detecting
Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) during the
dry season in Belo Horizonte, Minas Gerais, Brazil. Neotrop.
Entomol. 36: 294-302.
Gubler, D.J. 1998. Dengue and dengue hemorrhagic fever. Clin.
Microbiol. Rev. 3: 48–96.
Gubler, D.J., P. Reiter, K.L Ebi, W. Yap, R. Nasci, and J.A. Patz. 2001.
Climate variability and change in the United States: potential
impacts on vector- and rodent-borne diseases. Environ. Hlth
.Perspect. 2: 222-233.
Honório, N.A., C.T. Codeço, F.C. Alves, M.A.F.M. Magalhães,
and R. Lourenço-De-Oliveira. 2009. Temporal distribution of
Aedes aegypti in different districts of Rio de Janeiro, Brazil,
measured by two types of traps. J. Med. Entomol. 46: 1001–
1014.
Joshi, V., D.T. Mourya, and R.C. Sharma. 2002. Persistence of
dengue-3 virus through transovarial transmission passage in
successive generations of Aedes aegypti mosquitoes. Am. J.
Trop. Med. Hyg. 67: 158-161.
Khan, M.I.H., E. Anwar, A. Agha, N.S.M. Hassanien, E. Ullah, I.A.
Syed, and A. Raja. 2013. Factors predicting severe dengue in
patients with dengue fever. Medit. J. Hematol. Infect. Dis. 5:
e2013014.
Kow, C.Y., L.L. Koon, and P.F. Yin. 2001. Detection of dengue
viruses in field caught male Aedes aegypti and Aedes albopictus
(Diptera:Culicidae) in Singapore by type–specific PCR. J.
Med. Entomol. 38: 475-479.
Miyazaki, R.D., A.L.M. Ribeiro, M.G. Pignatti, and J.H. Campelo
Jr. 2009. Monitoring of Aedes aegypti mosquitoes (Linnaeus,
1762) (Diptera: Culicidae) by means of ovitraps at the
Universidade Federal de Mato Grosso Campus, Cuiabá, State
of Mato Grosso. Rev. Soc. Bras. Med. Trop. 42: 392-397.
Morin, C.W., A.C. Comrie, and K. Ernst. 2013. Climate and
dengue transmission: evidence and implications. Environ.
Hlth. Perspect. 121: 1264-1272.
Nunes, L.S., R.B.R. Trindade, and R.N.P. Souto. 2011. Avaliação
da atratividade de ovitrampas a Aedes (Stegomyia) aegypti
Linneus (Diptera: Culicidae) no bairro Hospitalidade,
Santana, Amapá. Bio. Amaz. 1: 26-31.
Pessanha, J.E.M., W.T. Caiaffa, A.B. Cecilio, F.C.M. Iani, S.C.
Araujo, J.C. Nascimento, E.G. Kroon, F.A. Proietti, and J.R
Arias. 2011. Cocirculation of two dengue virus serotypes in
individual and pooled samples of Aedes aegypti and Aedes
albopictus larvae. Rev. Soc. Bras. Med. Trop. 44: 103-105.
Primavesi, R. 2013. Wedding fever: fever in the returning traveler.
Diagnosis: Dengue fever. Can. Fam. Phys. 59: 742-744.
Regis, L.N., R.V Acioli, J.C.S. Júnior, M.A.V. Melo-Santos, W.V.
Souza, C.M. Ribeiro, J.C. da Silva, A.M. Monteiro, C.M.
Oliveira, R.M. Barbosa, C. Braga, M.A. Rodrigues, M.G.
Silva, P.J. Ribeiro, W.H. Bonat Jr., L.C. de Castro Medeiros,
74 Journal of Vector Ecology June 2015
M.S. Carvalho, and A.F. Furtado. 2013. Sustained reduction
of the dengue vector population resulting from an integrated
control strategy applied in two Brazilian cities. PlosOne 8:
e67682.
Regis, L., A.M. Monteiro, M.A.V Melo-Santos, J.C. Silveira Jr.,
A.F. Furtado, R.V. Acioli, G.M. Santos, M. Nakazawa, M.S.
Carvalho, P.J. Ribeiro Jr., and W.V. Souza. 2008. Developing
new approaches for detecting and preventing Aedes aegypti
population outbreaks: basis for surveillance, alert and control
system. Mem. Inst. Oswaldo Cruz 3: 50-59.
Reiter, P. 2001. Climate change and mosquito-borne disease.
Environ. Hlth. Perspect. 109: 141-161.
Reiter, P., M.A. Amador, and C. Nelson. 1991. Enhancement of the
CDC ovitrap with hay infusions for daily monitoring of Aedes
aegypti populations. J. Am. Mosq. Contr. Assoc. 7: 52-55.
Resende, M.C., T.M. Azara, I.O. Costa, L.C. Heringer, M.R.
Andrade, J.L. Acebal, and A.E. Eiras. 2012. Field optimisation
of MosquiTRAP sampling for monitoring Aedes aegypti
Linnaeus (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz 107:
294-302.
Resende, M.C., I.M. Silva, B. Ellis, and A.E. Eiras. 2013. A
comparison of larval, ovitrap and MosquiTRAP surveillance
for Aedes (Stegomyia) aegypti. Mem. Inst. Oswaldo Cruz 108:
1024-1030.
Restrepo, B.N., M.E. Beatty, Y. Goez, R.E. Ramirez, G.W. Letson,
F.J. Diaz, L.D. Piedrahita, and J.E. Osorio. 2014. Frequency
and clinical manifestations of dengue in urban Medellin,
Colombia. J. Trop. Med. 2014: 1-8.
Russell, R.C. 1998. Mosquito-borne arboviruses in Australia: the
current scene and implications of climate change for human
health. Int. J. Parasitol. 28: 955-969.
Santos, S.R.A., M.A.V. Melo-Santos, L. Regis, and C.M.R.
Albuquerque. 2003. Field evaluation of ovitraps consociated
with grass infusion and Bacillus thuringiensis var. israelensis
to determine oviposition rates of Aedes aegypti. Dengue Bull.
27: 156-162.
Vélez, I.D., M.L. Quiñones, M. Suárez, V. Olano, L.M. Murcia, E.
Correa, C. Arévalo, L. Pérez, H. Brochero, and A. Morales.
1998. A presencia de Aedes albopictus en Leticia, Amazonas,
Colombia. Biomédica 18: 192-198.
Zeidler, J.D., P.O.A. Acosta, P.P. Barrêto, and J.S. Cordeiro. 2008.
Vírus dengue em larvas de Aedes aegypti e sua dinâmica de
infestação, Roraima, Brasil. Rev. Saúde Públ. 42: 986-991.
Zell, R. 2004. Global climate change and the emergence/re-
emergence of infectious diseases. Int. J. Med. Microbiol. 293:
16-26.