Tropical Medicine and International Health
doi:10.1111/tmi.12373
volume 00 no 00
Oral susceptibility of Aedes aegypti (Diptera: Culicidae) from Senegal for dengue serotypes 1 and 3 viruses Alioune Gaye1,2, Oumar Faye3, Cheikh T. Diagne1,2, Ousmane Faye3, Diawo Diallo1, Scott C. Weaver4, Amadou A. Sall4 and Mawlouth Diallo1 1 2 3 4
Unite d’entomologie Medicale, Institut Pasteur de Dakar, Dakar, Senegal Universite Cheikh Anta Diop de Dakar, Dakar, Senegal Unite des Arbovirus et virus de Fievres Hemorragiques, Institut Pasteur de Dakar, Dakar, Senegal 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 RT–PCR to estimate the infection and dissemination rates. results The data indicated low susceptibility to DENV-3 (infection: 2.4–15.2%, and dissemination rates: 0–8.3%) 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 millions 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 different (DENV 1–4) 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 d’Ivoire 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 Senegal. Sylvatic DENV-2 amplifications 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
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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 (Amarasinghe 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 competence 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 populations of Ae. aegypti from Senegal for DENV-3 and DENV-1. Materials and methods Two populations of Ae. aegypti were used: a purely sylvatic and zoophilic population collected in the forest gallery of Kedougou (12°330 00″ N, 12°110 00″ W) breeding in tree holes and a domestic and anthropophilic population collected in the urban environment of Dakar (14°430 29″ N, 17°280 24″ W) breeding in artificial containers. Morphologically, the lack and presence of pale scales on the first abdominal tergite differentiate, 1
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Table 1 Chronological worldwide reports about vector competence of Aedes aegypti and Aedes albopictus for dengue 1 and 3 viruses Origin and history of the virus strains used
Origin of mosquitoes used
Serotypes
Country/host/year
Passage history
Species
Geographic origin
DENV1
Unknown
Unknown
Ae. albopictus
Unknown
Unknown
Ae. aegypti
Fiji/human/1975
1 C6/36
Ae. albopictus
Fiji/human/1975 Puerto Rico/human/1985 Puerto Rico/human/1985 Fiji/human/1975 Puerto Rico/human/1985
1 1 1 1 1
Ae. Ae. Ae. Ae. Ae.
Vietnam, Madagascar, Malaysia, India, Taiwan, Thailand, Hawaii, Mauritius, Indonesia, Philippine South Pacific, Indonesia, Malaysia, Singapore, Philippine, Thailand, Burma, Kenya, Burkina Malaysia, Japan, Texas, Tennessee, Louisiana Texas (USA) Houston (USA) Rexville (USA) Hawaii Brazil
Taiwan/human/1987
Ae. albopictus Ae. aegypti
Taiwan
Durban/human/1985 Australia/human/1990 Unknown Florida/human/2010
1 Toxo, 1LLCMK2 3 Vero, 1C6/36 3 Mice, 4 mosq 1 C6/36 Unknown 1 AGM, 2 Vero
Florida/human/2010
1 AGM, 2 Vero
Unknown
Unknown
Ae. aegypti Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypi
Mozambique/ human/1985 Mozambique/ human/1985 Unknown Philippines/human/1956 Thailand/human/1963 Unknown Cape Verde/ Human/2009
1 Mosq, 1 C3/36
DENV3
Test done
References
ST
Gubler & Rosen (1976)
ST
Gubler et al. (1979)
OT
Boromisa et al. (1987)
OT OT OT VT VT OT
Boromisa et al. (1987) Mitchell et al. (1987) Mitchell et al. (1987) Shroyer (1990) Mitchell & Miller (1990) Chen et al. (1993)
South Africa Australia China Florida (USA)
ST OT ST OT
Jupp and Kemp (1993) Watson & Kay (1999) Shu et al. (2004) Richards et al. (2012)
Florida (USA)
OT
Buckner et al. (2013)
OT
Gubler et al. (1979)
Ae. albopictus
South Pacific, Indonesia, Malaysia, Singapore, Thailand, Burma, Philippines, Kenya, Burkina Houston (USA)
OT
Mitchell et al. (1987)
1 Mosq, 1 C3/36
Ae. aegypi
Rexville (USA)
OT
Mitchell et al. (1987)
Unknown 1 Mice, 1 C6/36 21 Mouse Unknown 1 C6/36
Ae. Ae. Ae. Ae. Ae.
India Australia India China Cape Verde
VT ST VT ST OT
Joshi et al. (1996) Watson & Kay (1999) Joshi et al. (2002) Shu et al. (2004) Vazeille et al. (2013)
C6/36 Mosquito Mosquito C6/36, 2 Toxo Toxo
aegypti albopictus aegypti albopictus albopictus
aegypti aegypti aegypti albopictus aegypti
Toxo, toxorhynchites; Mosq, mosquito; OT, oral transmission; VT, vertical transmission; ST, susceptibility test.
respectively, the population from Kedougou related to Ae. aegypti formosus and Dakar compatible with Ae. aegypti 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 2
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 24–48 h, was exposed to artificial infectious blood meal for 30 min as previously described (Diallo et al. 2008). Fully engorged mosquitoes (n = 276) were selected and incubated at 27 1°C, 80 5% RH, and
© 2014 John Wiley & Sons Ltd
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A. Gaye et al. Susceptibility of Ae. aegypti to dengue
fed with 10% glucose. Samples of mosquitoes were collected 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 (50 ATTAGAGAGCAGATCTCTG 30 ) and reverse (50 TGACACGCGGTTTC 30 ), 0.5 ll of probe (50 TCAATATGCTGAAACGCG 30 ), 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 dissemination (number of infected legs – wings / number of DENV-positive bodies) rates were calculated. Fisher’s 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
significant 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 infections 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, respectively). 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 significant differences were observed between mosquito populations. The comparison of infection and dissemination rates, in the two Ae. aegypti populations for each DENV serotype and at each incubation period, did not reveal significant differences. The two Ae. aegypti populations showed higher infection and dissemination rates with DENV-1 compared with DENV-3. The differences were significant at 15 and 20 dpi for Ae. aegypti from Dakar (P < 0.009). Discussion Our findings revealed relatively low DENV-3 infection and dissemination rates in the two Senegalese populations of Ae. aegypti, compared with mosquitoes from Australia, 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 (0–12.5%) were obtained with Ae. aegypti from Burkina Faso and Kenya exposed to a virus titre ≥107.3 MID50/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 Infection and dissemination according to each extrinsic incubation period or day post-infection (dpi) Infection rate (%) Species Aedes aegypti (DKR) Aedes aegypti (KDG)
Virus strain DENV1_IbH28328 DENV3 H87 DENV1_IbH28328 DENV3 H87
Blood meal titre (MID50/ml) 3.3
5.10 5.104.4 5.104.3 5.104.2
7
15
0/9 4/40 (10) 2/5 (40) 0/5
14/32 12/79 3/10 1/12
Dissemination rate (%)
(43.7) (15.2) (30) (8.3)
20
7
15
20
4/13 (30.8) 1/41 (2.4) 3/6 (50) NT
NT 0/4 0/2 NT
1/14 (7.14) 1/12 (8.3) 0/3 0/1
2/4 (50) 0/1 1/3 (33.3) 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.
© 2014 John Wiley & Sons Ltd
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Manila and Singapore, which developed 16.9–18.7% infection rates with a virus titre ≥106.7 MID50/ml (Gubler et al. 1979), or Thai mosquitoes, where an infectious blood meal ≥107 MID50/ml generated 0.9–19.7% infection (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 findings revealed susceptibilities comparable with those described for Ae. aegypti from dengue-endemic locations (Gubler et al. 1979; Thongrungkiat et al. 2003; Vazeille et al. 2013). However, 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. aegypti 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 determine 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 d’Entomologie Medicale at Institut Pasteur de Dakar as well as our field 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, 1349–1354. Bhatt S, Gething PW, Brady OJ et al. (2013) The global distribution and burden of dengue. Nature 496, 504–507. 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, 378–386. Buckner EA, Alto BW & Lounibos LP (2013) Vertical transmission of Key West dengue-1 virus by Aedes aegypti and Aedes albopictus (Diptera: Culicidae) mosquitoes from Florida. Journal of Medical Entomology 50, 1291–1297.
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Chen WJ, Wei HL, Hsu EL & Chen ER (1993) Vector competence of Aedes albopictus and Ae. Aegypti (Diptera: Culicidae) to dengue 1 virus on Ta€ıwan: development of the virus in orally and parenterally infected mosquitoes. Journal of Medical Entomology 30, 524–530. Diallo M, Ba Y, Sall AA et al. (2003) Amplification of the sylvatic cycle of dengue virus type 2, Senegal, 1999–2000: entomologic findings and epidemiologic considerations. Emerging Infectious Diseases 9, 362–367. Diallo M, Ba Y, Faye O et al. (2008) Vector competence of Aedes aegypti populations from Senegal for sylvatic and epidemic dengue 2 virus isolated in West Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 102, 493–498. Faye O, Ba Y, Faye O et al. (2014) Major urban dengue 3 epidemic in Senegal in 2009. Emerging Infectious Diseases 20, 456–459. Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clinical Microbiology Reviews 11, 480–496. 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, 318–325. 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, 1045–1052. Joshi V, Singhi M & Chaudhary RC (1996) Transovarial transmission 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 dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. American Journal of Tropical Medicine and Hygiene 67, 158–161. 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, 639–643. 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, 251–253. Mitchell CJ, Miller BR & Gubler DJ (1987) Vector competence of Aedes albopictus from Houston, Texas, for dengue serotypes 1 to 4, yellow fever and Ross River viruses. Journal of the American Mosquito Control Association 3, 460–465. Richards SL, Anderson SL & Alto BW (2012) Vector competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for dengue virus in the Florida Keys. Journal of Medical Entomology 49, 942–946. 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. 1974–1985. Bulletin de la Societe de Pathologie Exotique et de ses Filiales 79, 313–322.
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Sanchez-Vargas I, Scott JC, Poole-Smith BK et al. (2009) Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito’s 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, 312–314. Shu LP, Zuo L, Zhao X, Chen AY & Wei LH (2004) Susceptibility 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, 234–237. 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. Journal of Vector Ecology: Journal of the Society for Vector Ecology 28, 166–170. Vazeille M, Yebakima A, Lourencßo-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, 37–40. Watson TM & Kay BH (1999) Vector competence of Aedes notoscriptus (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, 508–514.
Corresponding Author Mawlouth Diallo, Unite d’Entomologie Medicale, Institut Pasteur de Dakar, BP 220 Dakar, Senegal. Tel.: +221.338399228; Fax +221.338399210; E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
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doi:10.1111/tmi.12373
volume 00 no 00
Oral susceptibility of Aedes aegypti (Diptera: Culicidae) from Senegal for dengue serotypes 1 and 3 viruses Alioune Gaye1,2, Oumar Faye3, Cheikh T. Diagne1,2, Ousmane Faye3, Diawo Diallo1, Scott C. Weaver4, Amadou A. Sall4 and Mawlouth Diallo1 1 2 3 4
Unite d’entomologie Medicale, Institut Pasteur de Dakar, Dakar, Senegal Universite Cheikh Anta Diop de Dakar, Dakar, Senegal Unite des Arbovirus et virus de Fievres Hemorragiques, Institut Pasteur de Dakar, Dakar, Senegal 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 RT–PCR to estimate the infection and dissemination rates. results The data indicated low susceptibility to DENV-3 (infection: 2.4–15.2%, and dissemination rates: 0–8.3%) 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 millions 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 different (DENV 1–4) 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 d’Ivoire 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 Senegal. Sylvatic DENV-2 amplifications 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
© 2014 John Wiley & Sons Ltd
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 (Amarasinghe 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 competence 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 populations of Ae. aegypti from Senegal for DENV-3 and DENV-1. Materials and methods Two populations of Ae. aegypti were used: a purely sylvatic and zoophilic population collected in the forest gallery of Kedougou (12°330 00″ N, 12°110 00″ W) breeding in tree holes and a domestic and anthropophilic population collected in the urban environment of Dakar (14°430 29″ N, 17°280 24″ W) breeding in artificial containers. Morphologically, the lack and presence of pale scales on the first abdominal tergite differentiate, 1
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Table 1 Chronological worldwide reports about vector competence of Aedes aegypti and Aedes albopictus for dengue 1 and 3 viruses Origin and history of the virus strains used
Origin of mosquitoes used
Serotypes
Country/host/year
Passage history
Species
Geographic origin
DENV1
Unknown
Unknown
Ae. albopictus
Unknown
Unknown
Ae. aegypti
Fiji/human/1975
1 C6/36
Ae. albopictus
Fiji/human/1975 Puerto Rico/human/1985 Puerto Rico/human/1985 Fiji/human/1975 Puerto Rico/human/1985
1 1 1 1 1
Ae. Ae. Ae. Ae. Ae.
Vietnam, Madagascar, Malaysia, India, Taiwan, Thailand, Hawaii, Mauritius, Indonesia, Philippine South Pacific, Indonesia, Malaysia, Singapore, Philippine, Thailand, Burma, Kenya, Burkina Malaysia, Japan, Texas, Tennessee, Louisiana Texas (USA) Houston (USA) Rexville (USA) Hawaii Brazil
Taiwan/human/1987
Ae. albopictus Ae. aegypti
Taiwan
Durban/human/1985 Australia/human/1990 Unknown Florida/human/2010
1 Toxo, 1LLCMK2 3 Vero, 1C6/36 3 Mice, 4 mosq 1 C6/36 Unknown 1 AGM, 2 Vero
Florida/human/2010
1 AGM, 2 Vero
Unknown
Unknown
Ae. aegypti Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypi
Mozambique/ human/1985 Mozambique/ human/1985 Unknown Philippines/human/1956 Thailand/human/1963 Unknown Cape Verde/ Human/2009
1 Mosq, 1 C3/36
DENV3
Test done
References
ST
Gubler & Rosen (1976)
ST
Gubler et al. (1979)
OT
Boromisa et al. (1987)
OT OT OT VT VT OT
Boromisa et al. (1987) Mitchell et al. (1987) Mitchell et al. (1987) Shroyer (1990) Mitchell & Miller (1990) Chen et al. (1993)
South Africa Australia China Florida (USA)
ST OT ST OT
Jupp and Kemp (1993) Watson & Kay (1999) Shu et al. (2004) Richards et al. (2012)
Florida (USA)
OT
Buckner et al. (2013)
OT
Gubler et al. (1979)
Ae. albopictus
South Pacific, Indonesia, Malaysia, Singapore, Thailand, Burma, Philippines, Kenya, Burkina Houston (USA)
OT
Mitchell et al. (1987)
1 Mosq, 1 C3/36
Ae. aegypi
Rexville (USA)
OT
Mitchell et al. (1987)
Unknown 1 Mice, 1 C6/36 21 Mouse Unknown 1 C6/36
Ae. Ae. Ae. Ae. Ae.
India Australia India China Cape Verde
VT ST VT ST OT
Joshi et al. (1996) Watson & Kay (1999) Joshi et al. (2002) Shu et al. (2004) Vazeille et al. (2013)
C6/36 Mosquito Mosquito C6/36, 2 Toxo Toxo
aegypti albopictus aegypti albopictus albopictus
aegypti aegypti aegypti albopictus aegypti
Toxo, toxorhynchites; Mosq, mosquito; OT, oral transmission; VT, vertical transmission; ST, susceptibility test.
respectively, the population from Kedougou related to Ae. aegypti formosus and Dakar compatible with Ae. aegypti 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 2
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 24–48 h, was exposed to artificial infectious blood meal for 30 min as previously described (Diallo et al. 2008). Fully engorged mosquitoes (n = 276) were selected and incubated at 27 1°C, 80 5% RH, and
© 2014 John Wiley & Sons Ltd
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fed with 10% glucose. Samples of mosquitoes were collected 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 (50 ATTAGAGAGCAGATCTCTG 30 ) and reverse (50 TGACACGCGGTTTC 30 ), 0.5 ll of probe (50 TCAATATGCTGAAACGCG 30 ), 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 dissemination (number of infected legs – wings / number of DENV-positive bodies) rates were calculated. Fisher’s 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
significant 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 infections 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, respectively). 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 significant differences were observed between mosquito populations. The comparison of infection and dissemination rates, in the two Ae. aegypti populations for each DENV serotype and at each incubation period, did not reveal significant differences. The two Ae. aegypti populations showed higher infection and dissemination rates with DENV-1 compared with DENV-3. The differences were significant at 15 and 20 dpi for Ae. aegypti from Dakar (P < 0.009). Discussion Our findings revealed relatively low DENV-3 infection and dissemination rates in the two Senegalese populations of Ae. aegypti, compared with mosquitoes from Australia, 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 (0–12.5%) were obtained with Ae. aegypti from Burkina Faso and Kenya exposed to a virus titre ≥107.3 MID50/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 Infection and dissemination according to each extrinsic incubation period or day post-infection (dpi) Infection rate (%) Species Aedes aegypti (DKR) Aedes aegypti (KDG)
Virus strain DENV1_IbH28328 DENV3 H87 DENV1_IbH28328 DENV3 H87
Blood meal titre (MID50/ml) 3.3
5.10 5.104.4 5.104.3 5.104.2
7
15
0/9 4/40 (10) 2/5 (40) 0/5
14/32 12/79 3/10 1/12
Dissemination rate (%)
(43.7) (15.2) (30) (8.3)
20
7
15
20
4/13 (30.8) 1/41 (2.4) 3/6 (50) NT
NT 0/4 0/2 NT
1/14 (7.14) 1/12 (8.3) 0/3 0/1
2/4 (50) 0/1 1/3 (33.3) 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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Manila and Singapore, which developed 16.9–18.7% infection rates with a virus titre ≥106.7 MID50/ml (Gubler et al. 1979), or Thai mosquitoes, where an infectious blood meal ≥107 MID50/ml generated 0.9–19.7% infection (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 findings revealed susceptibilities comparable with those described for Ae. aegypti from dengue-endemic locations (Gubler et al. 1979; Thongrungkiat et al. 2003; Vazeille et al. 2013). However, 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. aegypti 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 determine 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 d’Entomologie Medicale at Institut Pasteur de Dakar as well as our field 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, 1349–1354. Bhatt S, Gething PW, Brady OJ et al. (2013) The global distribution and burden of dengue. Nature 496, 504–507. 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, 378–386. Buckner EA, Alto BW & Lounibos LP (2013) Vertical transmission of Key West dengue-1 virus by Aedes aegypti and Aedes albopictus (Diptera: Culicidae) mosquitoes from Florida. Journal of Medical Entomology 50, 1291–1297.
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Chen WJ, Wei HL, Hsu EL & Chen ER (1993) Vector competence of Aedes albopictus and Ae. Aegypti (Diptera: Culicidae) to dengue 1 virus on Ta€ıwan: development of the virus in orally and parenterally infected mosquitoes. Journal of Medical Entomology 30, 524–530. Diallo M, Ba Y, Sall AA et al. (2003) Amplification of the sylvatic cycle of dengue virus type 2, Senegal, 1999–2000: entomologic findings and epidemiologic considerations. Emerging Infectious Diseases 9, 362–367. Diallo M, Ba Y, Faye O et al. (2008) Vector competence of Aedes aegypti populations from Senegal for sylvatic and epidemic dengue 2 virus isolated in West Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 102, 493–498. Faye O, Ba Y, Faye O et al. (2014) Major urban dengue 3 epidemic in Senegal in 2009. Emerging Infectious Diseases 20, 456–459. Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clinical Microbiology Reviews 11, 480–496. 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, 318–325. 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, 1045–1052. Joshi V, Singhi M & Chaudhary RC (1996) Transovarial transmission 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 dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. American Journal of Tropical Medicine and Hygiene 67, 158–161. 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, 639–643. 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, 251–253. Mitchell CJ, Miller BR & Gubler DJ (1987) Vector competence of Aedes albopictus from Houston, Texas, for dengue serotypes 1 to 4, yellow fever and Ross River viruses. Journal of the American Mosquito Control Association 3, 460–465. Richards SL, Anderson SL & Alto BW (2012) Vector competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for dengue virus in the Florida Keys. Journal of Medical Entomology 49, 942–946. 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. 1974–1985. Bulletin de la Societe de Pathologie Exotique et de ses Filiales 79, 313–322.
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Sanchez-Vargas I, Scott JC, Poole-Smith BK et al. (2009) Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito’s 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, 312–314. Shu LP, Zuo L, Zhao X, Chen AY & Wei LH (2004) Susceptibility 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, 234–237. 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. Journal of Vector Ecology: Journal of the Society for Vector Ecology 28, 166–170. Vazeille M, Yebakima A, Lourencßo-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, 37–40. Watson TM & Kay BH (1999) Vector competence of Aedes notoscriptus (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, 508–514.
Corresponding Author Mawlouth Diallo, Unite d’Entomologie Medicale, Institut Pasteur de Dakar, BP 220 Dakar, Senegal. Tel.: +221.338399228; Fax +221.338399210; E-mail: [email protected]
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