Zika virus in southeastern Senegal: survival of the vectors and the virus during the dry season

Zika virus (ZIKV, genus Flavivirus, family Flaviviridae) was isolated for the first time in the Zika forest near Entebbe, Uganda, in 1947 from a febrile sentinel Rhesus monkey (Macaca mulatta). One year later the virus was isolated from Aedes africanus in the same area [1]. The first human case was reported in 1952 in Nigeria [2]. Subsequently, serological and virological studies showed the circulation of ZIKV in several other African [3] and Asian countries [4]. The virus was almost silent for 60 years until its reemergence in 2007 with the Yap Island and Gabon outbreaks [5]. It is during the last decade that the Asian genotype of ZIKV has experienced significant geographic expansion causing major epidemics in the Pacific Islands [6] and in America [7]. Indeed, phylogenic studies on sequenced ZIKV strains isolated from Brazil, Puerto Rico and Guatemala indicated that they were all within the Asian genotype and closely related to the French Polynesia strain [8, 9]. ZIKV is known to cause several debilitating neurological complications, including microcephaly in newborns and Guillain Barré Syndrome in adults, described in both Latin America and the Pacific Islands [10‐13]. In the urban cycle, ZIKV is mainly transmitted by Ae. aegypti and Ae. albopictus, but cases of non-vector transmission like sexual transmission, blood transfusion, or other fluid transmissions were reported [7, 14, 15]. In West Africa, ZIKV has a sylvatic cycle (described in southeastern Senegal) involving mainly non-human primates and arboreal Aedes (Ae. furcifer, Ae. luteocephalus, Ae. africanus, Ae. vittatus, Ae. dalzieli and Ae. taylori), while Ae. africanus plays this role in Central and Eastern Africa [16, 17]. In this cycle, humans are rarely infected when entering in forest or by Ae. furcifer and Ae. vittatus in villages [17].

Evidences of ZIKV presence in human populations in Senegal were shown by the detection of the virus by serological and virological tests in samples collected from several localities (Dakar, Casamance, Ferlo, Diourbel, Sine Saloum, southeastern Senegal, Bandia, and Meckhé) between 1962 and 2015 [17‐20]. In vectors, ZIKV was isolated in western Senegal from Ae. luteocephalus collected in 1968 in the Saboya forest [21] and from three mosquito species (Aedes luteocephalus, Aedes furcifer-taylori and Anopheles gambiae) in Bandia. As part of a program to study the biodiversity of arboviruses and their vectors in southeastern Senegal (1972–2015), more than 400 strains of ZIKV were isolated from around 20 species of mosquitoes belonging to the genera Aedes, Anopheles, Culex and Mansonia [17]. Recent studies have detected ZIKV from mosquitoes collected in different types of land covers including forests, savannahs, barrens, agricultures and villages across a large geographical area indicating wide spread of ZIKV in southeastern Senegal [17]. However, the main vectors belong to the genera Aedes (subgenera Aedimorphus (Ae. dalzieli), Diceromyia (Ae. furcifer, Ae. taylori) and Stegomyia (Ae. luteocephalus)) accounting for more than 90% of the isolates.

ZIKV is the most frequently amplified arbovirus in Senegal [12, 17, 21, 22]. Indeed, it was detected in mosquitoes during 21 of the 44 years of monitoring between 1972 and 2015 in the southeastern part of the country. However, ZIKV prevalence and vector density drop dramatically beyond detection by current methods each year during the dry season, generally between January and May. These data suggest that in southeastern Senegal, vectors and ZIKV are maintained locally in nature during adverse conditions corresponding to the dry season and the years when the virus is undetectable by current methods. Since the vectors belong mainly to the Aedes genus, their maintenance in nature is probably done through their desiccation-resistant eggs in larval breeding sites. The presence of eggs of 9 Aedes species from dry tree holes from one forest-gallery has already been demonstrated by Diallo and others in 1999–2000 [23]. However, the relative importance of tree holes for the dry season maintenance of these Aedes eggs compared to the others breeding sites [24] has not yet been investigated. The same is true of forests compared to other land covers. The detection of the virus in a pool of male Ae. furcifer in 2011 [17] suggests that vertical transmission, from infected females to its progeny via eggs, may be an important mechanism for local virus maintenance. In addition, there is currently no data on the mechanisms and vertical transmission rates in nature. There is also no data on species and breeding sites involved and their levels of participation in this mechanism. Our first objective was to identify the vector species in which Zika virus is maintained during dry season, and their specific infection rates, using genetic testing methods. Our second objective was to identify if vector species are more likely to use certain combinations of breeding site types and land cover classes than others to lay their eggs, using mainly a qualitative statistical approach on vector species occurrence. We expected that Zika virus would be maintained through certain mosquito species, and these mosquito species preferentially use certain combinations of breeding site types and land cover classes to lay their eggs.

Our study showed that 13 Aedes species eggs were maintained in the different land covers and breeding sites sampled in southeastern Senegal. This diversity is greater than that observed in a previous study conducted in the same area with only 9 Aedes species emerged from eggs samples collected in tree holes [23]. The higher number of breeding sites and land cover classes investigated in our study would explain this difference. Indeed, Diallo and others [23] only surveyed tree holes from a single forest gallery while our study covered 4 types of breeding sites in 5 different land cover classes. However, 12 Aedes species previously found as larvae [24] or adults [27] in this area were not found in this study. This could be explained by the inaccessibility of tree holes located at height higher than 2 m, which may be preferential habitat for these arboreal species. Indeed, variations of breeding behavior of some mosquito species depending on tree height were observed by some authors [34, 35]. The entrances of these tree holes may also be hidden by their small sizes or their location behind tree barks. Despite the fact that we were interested to the eggs (that are not mobile), sampling in different months and years could be considered as a limitation in our study.

Our study also showed that fruit husks were not involved in vectors maintenance during the dry season. This result was not expected because between 89 and 100% of these fruit husks were positives for larvae or pupae of 8 Aedes species in a previous study in the same area [24]. The particular physical, chemical and biological conditions of water contained in these fruit husks could alter the potential of resistance of Aedes eggs to desiccation resulting in the destruction of all eggs that did not hatch during rainy season. Further investigations on conditions that prevent fruit husks from being used as Aedes eggs maintenance sites could ultimately lead to the identification of substances or organisms that can be used for eggs destruction, and thus, in controlling major arboviruses of medical importance such as Zika, dengue, yellow fever and chikungunya. Maintenance rates were quite low compared to larval presence rates during the rainy season for all vectors listed, indicating an impact of the climatic conditions on the survival of Aedes eggs [36]. Indeed, even if Aedes eggs are resistant to desiccation, their viability decreases with time. The low relative humidity of the air also reduces survival time of Aedes eggs [37, 38]. In addition, the proportion of resistant eggs during a given period is greater in shaded sites than those exposed to the sun [39].

The dominant species Ae. aegypti was present in almost all breeding sites and land covers sampled. The plasticity of this species observed in our study is consistent with larval survey data in the area and elsewhere [24, 40] but also with its oviposition behavior in Ivory Coast [41]. This greater plasticity of Ae. aegypti suggests that its eggs are more resistant to desiccation and lower humidity than other species [37, 42].

Finding viable eggs of Ae. taylori maintained in villages was not expected. Indeed, this species was almost never found biting humans within villages area [17, 26]. This species could feed on other animals within villages or breed in some villages close to wild environment before going back to forests and savannahs where it bites. The localized maintenance of Ae. furcifer in tree holes from forests is consistent with its oviposition behavior but discordant with the fact that it could feed in all the land covers found in the area including villages indoors [17].

Viable eggs of Ae. vittatus were maintained in rock holes of forests in concordance with its breeding preference in Nigeria [43]. Nevertheless, larval survey data in the area and elsewhere have shown that the species can colonize a wide range of breeding sites and land cover classes [24, 44]. The maintenance of Ae. vittatus exclusively in rock holes from forests could be explained by total egg mortality in other land covers that are exposed to very high temperatures associated with low relative humidity. Rock holes from forests in which Ae. vittatus was maintained were relatively shady and filled with dead leaves creating a microclimate that is less warm, wetter and thus more favorable to eggs survival of this species.

ZIKV detection by RT-PCR in 4 mosquito pools from 3 different species indicating for the first time that the virus can be maintained locally during dry season in these potential vectors through vertical transmission. This possibility had already been suggested by detection of the virus from a male Ae. furcifer in the area [17]. A recent study detected ZIKV in 5 adults Ae. albopictus emerged from eggs collected in 2015 in Bahia, Brazil [45]. In addition, vertical transmission of ZIKV has also been proven for Ae. aegypti and Ae. albopictus in laboratory studies [46, 47]. The maintenance of ZIKV in 3 species in southeastern Senegal explains its frequent amplification from the beginning of the rainy season. Although these vertical transmission rates of arboviruses are generally low, they are of great epidemiological importance in nature. Indeed, they allow prolonged conservation of viruses during unfavorable periods to transmission (dry season) where vectors live in state of eggs and sensitive hosts are rare. The implication of Ae. vittatus in ZIKV maintenance strengthens its status of potential epidemic vector. Indeed, Ae. vittatus has already been found associated with ZIKV in nature and competent for the virus in laboratory [17, 48]. Our study revealed for the first time the association of Ae. bromeliae with ZIKV in the area. Particular attention should be paid to this vector belonging to the same group as Ae. simpsoni which is one of the main vectors of yellow fever in East Africa [49]. Nevertheless, further studies are needed on the vector competence of this species for ZIKV. Aedes unilineatus has already been found associated to ZIKV in nature and able to disseminate but not to transmit the virus [17, 48]. Its implication in maintaining ZIKV suggests that it may also play a more important epidemiological role in ZIKV epidemiology in Africa.

This study allows us to identify breeding sites, land cover classes and maintenance rates of several important ZIKV vectors in southeastern Senegal. It has also highlighted ZIKV maintenance in the area as well as species involved and breeding sites and land covers in which virus is maintained. These results provide a better understanding of the epidemiology of ZIKV disease in southeastern Senegal.