A Systematic Review: Is Aedes albopictus an Efficient Bridge Vector for Zoonotic Arboviruses?

Mosquito-borne arboviruses are increasing due to human disturbances of natural ecosystems and globalization of trade and travel. These anthropic changes may affect mosquito communities by modulating ecological traits that influence the "spill-over" dynamics of zoonotic pathogens, especially at the interface between natural and human environments. Particularly, the global invasion of Aedes albopictus is observed not only across urban and peri-urban settings, but also in newly invaded areas in natural settings. This could foster the interaction of Ae. albopictus with wildlife, including local reservoirs of enzootic arboviruses, with implications for the potential zoonotic transfer of pathogens. To evaluate the potential of Ae. albopictus as a bridge vector of arboviruses between wildlife and humans, we performed a bibliographic search and analysis focusing on three components: (1) The capacity of Ae. albopictus to exploit natural larval breeding sites, (2) the blood-feeding behaviour of Ae. albopictus, and (3) Ae. albopictus' vector competence for arboviruses. Our analysis confirms the potential of Ae. albopictus as a bridge vector based on its colonization of natural breeding sites in newly invaded areas, its opportunistic feeding behaviour together with the preference for human blood, and the competence to transmit 14 arboviruses.


Introduction
The human alteration of Earth's natural systems has become a great concern and a threat to human health. Indeed, these changes are likely to drive most of the global disease burden over the coming century [1]. During the last decades, the burden of emerging infectious diseases has increased to represent a substantial threat to global health, security, and economy growth. About 75% of emerging infectious diseases are zoonotic diseases, mostly of wildlife origin [2,3]. The risk of zoonotic emergences is considered high in tropical forest regions associated with a range of facilitating factors, particularly high vertebrate species diversity and agricultural land use changes [4]. Understanding the mechanisms of disease emergence allows the development of early detection and control programs for reducing disease incidence and economic burden [5].
Zoonotic pathogens can be transmitted from animals to humans directly, or indirectly when arthropod vectors are needed to accomplish their life cycle. Zoonotic vector-borne diseases are maintained in enzootic cycles, but can be transmitted from animal reservoir populations to sympatric human populations or to domestic animals during "spill-over events", and also from humans to animals during "spill-back events" [2,6]. The global emergence of vector-borne diseases is helped by international travel and trade, after their local emergence has been driven by a combination of environmental changes that are not yet completely understood [7]. Therefore, research is needed to water collection points (e.g., tree holes, bamboo stumps, or rock holes) as oviposition sites in the woods [24], particularly at the forest edge [24]. The capacity to colonize artificial man-made containers (together with desiccation-resistant and diapausing eggs) led to its "domestication". Its ecological plasticity to several habitats, its passive dispersion through the global transport of tires and inside cars [25], and the inefficiency of control programmes have allowed Ae. albopictus to become one of most invasive species worldwide [23,24]. In native and newly colonized areas, it has been found in urban, rural, and forest habitats; however, unclear information is available on its natural breeding sites and its presence in forested environments. Moreover, its presence in natural breeding sites in the invaded territories has not been analysed. In general, Ae. albopictus is considered an opportunistic feeder that is attracted to mammals, particularly humans, rather than other hosts [26,27]. However, to our knowledge, a detailed and quantified analysis of its host preferences has never been done.
In relation to epidemic virus transmission, Ae. albopictus has been considered the vector for the chikungunya virus (CHIKV), dengue virus (DENV), and ZIKAV in Gabon and Central Africa [28,29], for DENV and CHIKV in la Réunion island [30], and for CHIKV in Madagascar and Mayotte [30,31]. In Europe, it has been incriminated in Italy and France during CHIKV and DENV outbreaks [32,33] and in Japan in DENV transmission [34]. Moreover, this mosquito represents a potential risk of outbreaks in many other areas, for example, in Brazil and USA where Ae. albopictus is widespread [35][36][37][38]. Different studies have shown that Ae. albopictus can develop infection from up to 32 arboviruses [16,23,36]; however, to our knowledge, its ability to transmit any of them has not been clearly demonstrated yet.
In this work, we hypothesized that Ae. albopictus may have an active role as a bridge vector for the transfer from vertebrate hosts to humans (spill-over events) and therefore, in the emergence of enzootic arboviruses. To test this hypothesis, we reviewed and quantified: (1) Ae. albopictus' capacity to exploit natural water collections as larval breeding sites (as a proxy for its establishment in rural/sylvatic/forested areas) in native or invaded regions; (2) its feeding behaviour with regard to humans, domestic, or wild animals (as a proxy for the contact between vertebrate hosts and humans); and (3) its vector competence, tested experimentally for different arboviruses and natural infections reported from the field in mosquitoes (as a proxy for its potential for virus transmission in the field). Finally, we discuss the potential spill-over transmission risk from vertebrate hosts to humans and the methodological issues and knowledge gaps that need to be tackled.

Feeding Behaviour
Our quantification of the feeding behaviour indicates that Ae. albopictus has been mainly reported as a species that prefers mammals (including humans) as blood sources (mean and standard deviation: 92.0% ± 8), compared with birds (8% ± 8) and other animals (3.7% ± 1.7) [26,27,[63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] (Figure 2A). Among mammals, blood meals were mainly from humans (60%) than non-human species (30%). From an "interspecies risk of transfer" perspective, it is relevant to note that Ae. albopictus seems to be biting domestic animals (25%) more frequently than wildlife animals (10%) ( Figure 2B). Importantly, there is huge variability in the percentage of human blood meals in the different studies. Among domestic and peri-domestic animals, dogs, rodents, and rabbits were reported as the main blood sources for Ae. albopictus, followed by cats, bovines, chickens, horses, and pigs (Supplementary Figure S1). When classified according to the biological family of blood sources, Ae. albopictus can feed on 28 different host biological families, and preferentially on animals belonging to Hominidae (60%), Muridae (15%), Canidae (12%), and Phasianidae (10%) (see Table 1 for detailed information and Supplementary Table S3 for bibliographical information). Among domestic and peri-domestic animals, dogs, rodents, and rabbits were reported as the main blood sources for Ae. albopictus, followed by cats, bovines, chickens, horses, and pigs (Supplementary Figure S1). When classified according to the biological family of blood sources, Ae. albopictus can feed on 28 different host biological families, and preferentially on animals belonging to Hominidae (60%), Muridae (15%), Canidae (12%), and Phasianidae (10%) (see Table 1 for detailed information and Supplementary Table S3 for bibliographical information). The mean frequencies were calculated using the data found in articles that described different Ae. albopictus populations biting different animals in different locations. As these articles do not describe the same biological families, the total mean bite frequency does not correspond to 100%.
Among studies on Ae. albopictus vector competence, we found important variations concerning the methodology used to perform the infection (intra-thoracic inoculation of viruses, oral challenge using infected blood meals or infected animals), the mosquito strains, the viral strains and the virus loads used, the conditions of mosquito incubation (e.g., time, temperature), and the methods used to determine mosquito infection and transmission efficiency. Concerning the virus inoculation methodology, intra-thoracic injection was used for 11 viruses to assess vector infection, and oral infection was performed using infected hosts (n = 11 arboviruses), or membrane feeding methods (n = 11 arboviruses).

Discussion
In the present work, we tried to understand the potential role of the Asian tiger mosquito Ae. albopictus as a bridge vector that might favour the transfer of zoonotic arboviruses from enzootic or domestic hosts to humans and vice-versa. To this aim, we evaluated its ability to colonize natural breeding sites in newly invaded and native areas, its appetence for animal blood sources, and its global efficiency for transmitting arboviruses. This mosquito species was described as capable of developing infection from a large number of arboviruses in laboratory conditions [36]. However, based on the published evidences of vector competence, we found that transmission by Ae. albopictus is proven only for 14 of them, without considering the epidemic Aedes-borne CHIKV, DENV (4 serotypes), and ZIKAV.
In relation to the capacity of Ae. albopictus to establish in natural areas (rural/sylvan environments), tree holes were described as the most common natural breeding sites, although it has been detected also in bamboo stumps, and more sporadically in rock holes and plant axils [24]. Our analysis indicates that coconut shells, bromeliads, and bamboo stumps might be as common as tree holes, whereas rock holes and leaf axils of other plants are less frequently used. These results might be biased due to differences across studies related to sampling efforts and the environmental characteristics of sampled areas. Therefore, they should be confirmed by comparisons with larval sampling in natural and artificial breeding sites in natural areas and forest edges. Moreover, when possible, the productivity in these habitats should be described and compared by pupal sampling, with the same methodology used for quantifying the productivity of anthropic containers in urban areas [176]. For example, a study in Rio de Janeiro showed that the percentage of Ae. albopictus larvae in bromeliads corresponded to 0.18% of all sampled larva, demonstrating the low productivity of this breeding place [48]. However, studies describing the productivity of natural breeding sites in the natural environment or at an interface between natural and man-modified environments are lacking. In native forested areas, natural containers of larvae (tree holes, bamboo stumps, rock holes) were observed at the forest edge, like in a colonized forested area. Breeding sites in the deep forest have never been detected for this species [24,27].
Our results also confirmed the opportunistic feeding behaviour of Ae. albopictus and its strong preference for mammals, especially humans (humans = 60%, non-humans = 30%) compared with other groups, such as birds (4%). Ae. albopictus can feed on 28 different biological families. Reports on Ae. albopictus biting on any primates other than man were lacking until very recently. Specifically, a study described Ae. albopictus probing on a howler monkey that had just died due to YFV and was lying on the forest edge in Brazil [177]. This mosquito also bites domesticated animals-Muridae, Canidae, Phasianidae, Herpestidae, and Bovinae. Several studies suggested this opportunism. For instance, laboratory experiments on the host choice showed that this mosquito preferentially bites humans compared with other animals [30]. This opportunism was confirmed in studies on blood-fed mosquitoes collected in the field [27,30,69,78]. From our literature analysis, birds appeared as a non-preferential host group. Based on the reported proportion of blood meals, domestic and peri-domestic animals (25%) should be considered more relevant than wildlife (10%) as sources of zoonotic pathogens for Ae. albopictus. However, a limited number of studies were carried out in natural habitats where wildlife is abundant. Therefore, additional research is needed in natural areas to precisely describe the blood feeding patterns of Ae. albopictus and its interaction with wildlife. If possible, the availability of vertebrate hosts should be taken into account by using field census procedure and by calculating indexes of feeding preferences [178]. Such approaches should prevent the underestimation of the Ae. albopictus' potential to transmit pathogens from domestic/sylvatic vertebrate hosts to humans, but also from domestic to sylvatic vertebrate hosts, and vice versa. Our analysis also highlighted a huge variability in the proportion of human blood meals. This is a relevant factor for calculating the vector capacity, the disease reproduction rate (Ro), and the spill-over risk that may be determined by several parameters [178].
Comparing the vector competence of Ae. aegypti and Ae. albopictus for different epidemic viruses did not allow for a conclusion that there is a difference in their TE rates for ZIKV, CHIKV, DENV-1, and DENV-2. However, for bridge vectors*virus pairs, WNV TE was higher for Ae. albopictus than for Cx. pipiens, contrary to what was expected. Although the WNV transmission efficiency rate by Ae. albopictus is high in experimental conditions, this species has never been incriminated as a WNV vector in the field, possibly due to its low propensity to bite birds. Ae. albopictus presented similar TE rates as Hg. leucocelenus, a primary YFV vector within and at the edges of Brazilian forests [27,179]. However, few studies have been carried out to assess Hg. leucocelenus vector competence. In general, the contribution of laboratory studies for assessing the role of vector(s) in natural environments is limited.
Based on vector competence and blood meal studies, we conclude that Ae. albopictus could act as a bridge vector for many viruses (e.g., WNV, EEEV, ORUV, RRV, YFV, JEV, VEEV, LACV, RVFV, CVV, CHPV, JCV, and MAYV) with a potential risk for disease emergence. One of our goals was to identify in a quantitative way the viruses with a higher risk of emergence, and to develop an analysis to quantify the relative risk of transfer to humans of each enzootic arbovirus that can be efficiently transmitted by Ae. albopictus in laboratory conditions. The methodology used was based on two previous published works [180,181] that quantified the risk of WNV transfer by Culex mosquitoes. We then calculated the relative risk of Ae. albopictus-mediated virus transfer from its natural hosts to humans using a simplified version of Kilpatrick's equation (see Supplementary Information for more details concerning the methodology used and Figure S2) that takes into account Ae. albopictus vector competence for a given virus (i.e., TE), and the mean relative feeding frequencies on humans (FHi) and on animal hosts (FAi). Unfortunately, this analysis was hindered by the limited information available on the enzootic/sylvatic reservoirs of several of these arboviruses (some hosts remain unknown or are not sufficiently identified). Moreover, some viruses have many potential reservoirs, and their objective weighting is difficult. Additionally, data on Ae. albopictus propensity to bite a given animal reservoir species are often lacking (e.g., primates). Consequently, only biting frequencies at animal family levels could be used, leading to overly unreliable and speculative risk transfer estimates. Therefore, we chose not to include them here, although these estimates are crucial to better assess the risk of spill-over and emergence of enzootic arboviruses in relation with the secondary invasion of Ae. albopictus in forested areas.
Another important limitation of the present work is the great methodological variation and the lack of standardization of the protocols used to assess the vector competence of Ae. albopictus. Vector competence for arboviruses is influenced by genetic factors in the mosquito population and in the virus strain, such as the geographical genetic origin of the vector population or the interaction between the vector and arbovirus genotype [182,183]. Therefore, the intraspecific genetic variability in mosquito species/populations, as well as the intra-and inter-specific variability of arboviruses can affect vector competence and risk estimations. External factors, such as the incubation temperature, can also affect vector competence, and consequently the transmission and analysis of the risk [184].
Other factors interfering with the vector competence results are the way of ingesting the virus-infected blood (in vivo or in vitro), the viral load concentration, and the sensibility of the method used to detect the virus in the mosquito body or saliva. We are aware that our study is limited due to the methodological differences of the analysed articles, and also because the risk of arbovirus emergence is a multifactorial process and it is actually impossible to estimate the interactions of all factors with the limited evidences available. Thus, more standardized studies of vector competence and blood feeding preferences are necessary. In this sense, the project Infravec2 (https://infravec2.eu) is an important international initiative, and one of its themes is the standardization of methods.
In conclusion, data from the literature show that Ae. albopictus can colonize forest environments, and has possible interactions with domestic animals and wildlife, suggesting a risk for interaction with animal viruses. Such a risk is particularly high in areas that are considered to be biodiversity hotspots, such as the Congo and Amazon Basin forests. The presence of Ae. albopictus in small towns and hamlets in the Amazon Forest highlights the risk of spill-over of some arboviruses that cause human diseases, such as OROV, YFV, and MAYV [27]. In Brazil, Ae. albopictus populations are experimentally competent for YFV transmission, but this has not been confirmed by infecting Ae. albopictus [127,185]. In Africa, many arboviruses could be investigated to elucidate their potential transmission and emergence facilitated by Ae. albopictus, as done for CHIKV [152]. In the United States, where this mosquito species is widespread, its potential role in LACV, EEEV, WNV, and POTV transmission must be investigated [36,133,135]. In Asia and Oceania, the potential for inter-species transmission of JEV and RRV must be evaluated. It is important to take into account that the risk of arbovirus emergence is dynamic and in continuous evolution because mosquito populations, virus genetics, and the possibility of their contact varies according to time and place, and adaptations could be expected, particularly for invasive pathogens and vectors [186]. For instance, in the Indian Ocean region, the interaction between Ae. albopictus and CHIKV led to the selection of a virus strain that infects vectors and can spread around the world more easily. Studies on mutation selection for more susceptible arbovirus strains are still limited, but can be useful for predicting spill-over events [187]. Also, vector competence must be evaluated with as many strains as possible to maximize viral diversity, if possible using strains recently isolated from animals.
Our literature review showed that Ae. albopictus is competent for many different arboviruses, is present in natural habitats and forest edges, and can feed on several animal groups [30]. All these features make of Ae. albopictus a potential bridge vector of several emerging arboviruses (at least 14 viruses [23,36]), thus increasing the risk of spill-over and spill-back events. We hope that our approach will encourage more research to disentangle this risk in the field and the laboratory, with the aim of preventing the emergence of zoonotic diseases and reducing potential health and economic burdens, particularly for vulnerable populations.

Natural Breeding Sites
First, a literature search was done in Google Scholar to identify articles reporting the presence of Ae. albopictus in natural larval breeding sites and their types, using the keywords "Natural Breeding sites Aedes albopictus" or "Oviposition sites Aedes albopictus" or "Larval habitats Aedes albopictus". This allowed for the identification of 16 articles [43,44,46,52,54,55,61,62,[188][189][190][191][192][193][194] (Supplementary Table S1). From these articles, the main natural breeding sites were listed: bamboo stumps, bromeliads, coconut shells, leaf axils, rock holes, tree holes, snail shells, cacao shells, puddles, dead cow horns, dead leaves, ground cavity, hollow log, palm bracts, and palm leaves. Then, a search on each type of natural breeding site was carried out using PubMed, using the following words: (Aedes albopictus [Title/Abstract] AND "Breeding type" [Title/Abstract]). The aim of this search was to quantify the number of articles and the number of detections that described the presence of this mosquito in each of the identified natural breeding sites (Supplementary Table S2). Articles that did not quantify the number of times the breeding sites were found positive were excluded. The bibliographic search was done between August and December 2018.

Feeding Behaviour
A literature search was done in Google Scholar with the key words "blood meal" and "host feeding", followed by "Aedes albopictus" until December 2018. Three studies were excluded because they were considered unreliable: (i) the study by Gingrich and Williams, 2005 [67], which did not test for human blood meals, thus bringing a potential bias into the results; (ii) the study performed in a zoo by Tuten et al., 2012 [195]; and (iii) the study by Hess et al., 1968 [196] that was exclusively carried out in a bird area on Hawaii Island. Finally, 22 studies were selected (see references and details for each of them in Supplementary Table S3) to build a database of blood feeding preferences, based on the Ae. albopictus biting frequency for each host species, biological family, or group of vertebrate hosts (human, mammals, birds, domestic animals, wild animals). The database was used to quantify the relative importance as a blood meal of each host group and of specific hosts, based on the reported blood meal sources identified using different techniques (DNA sequencing, ELISA blond meal analyses, agarose gel precipitin). Then, these preferences were analysed independently of the host availability, which was quantified in very few studies.

Arbovirus Transmission
First, all referenced arboviruses that might be transmitted by Ae. albopictus were selected using the arbocat database from Centers for Disease Control and Prevention (CDC) (https://wwwn.cdc.gov/ arbocat/VirusBrowser.aspx). Then, Google Scholar and PubMed were searched with the key words "Virus name" and "Vector Competence", followed by "Aedes albopictus". Among the 49 articles obtained with this search, articles containing data on virus detection/isolation from field-collected mosquitoes, and data on vector competence parameters, including "susceptibility", "infection, dissemination", or "transmission rates" were selected (see Supplementary Table S4 showing the viruses and the bibliographic references). Data from each article were used to calculate the infection rates as the number of mosquitoes showing virus infection in the gut divided by the number of mosquitoes fed with infected blood x 100. Dissemination efficiency was calculated as the number of mosquitoes with viruses disseminated in the legs, wings, or head divided by the number of mosquitoes fed with infected blood x 100. Transmission rates were calculated as the number of mosquitoes that could deliver the virus with saliva (detection of virus in mosquito saliva, or demonstration of transmission using animal hosts exposed to infected mosquito bites) divided by the number of mosquitoes with viruses disseminated in the legs, wings, or head (body) × 100. Transmission efficiency was calculated as the number of mosquitoes that could deliver the virus with saliva (detection of living viruses or viral genome in mosquito saliva, or demonstration of transmission using animal hosts exposed to infected mosquito bites) divided by the number of mosquitoes fed with infected blood [168]. In the present work, infection performed from intrathoracic assays corresponds to mosquitoes that after intrathoracic injection, were detected with the virus after a 7-10 day incubation period. For this detection, the ground mosquito suspension was inoculated in rats, or the presence of the virus quantified by assays in Vero cell cultures. After intrathoracic injection, infected mosquitoes may transmit the virus to another animal. Some articles only described transovarial transmission tested after intrathoracic infection. These works demonstrated Ae. albopictus susceptibility to develop infection by a given arbovirus. However, these articles did not quantify the infection and transmission rates.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-0817/9/4/266/s1, Table S1. List of the 16 articles found by searching Google Scholar to characterize the types of natural breeding sites exploited by Ae. albopictus, Table S2. Typology and number of reported natural containers exploited by Ae. albopictus from articles found in PubMed, Table S3: List of references used to analyse the host feeding preferences of Aedes albopictus, Table S4: List of references that reported infection, infections rate, dissemination rate, dissemination efficiency, transmissions rate or transmission efficiency in Ae. albopictus for the indicated arboviruses, Table S5: List of references used to analyse the vector competence of several mosquito-virus pairs: Aedes aegypti*CHIKV, Aedes aegypti*DENV-1, Aedes aegypti*DENV-2, Aedes aegypti*ZIKV, Aedes albopictus*CHIKV, Aedes albopictus*DENV-1, Aedes albopictus*DENV_2, Aedes albopictus*ZIKV, Culex pipiens*WNV, and Haemagogus leucocelenus*YFV, Table S6 Table S7: Geographic distribution, vertebrate hosts and potential vectors of arboviruses isolated or tested for vector competence in Ae. albopictus. Figure S1. Analysis of the host feeding patterns of Ae. albopictus for the different species of domestic animals without taking into account the host availability.

Conflicts of Interest:
The authors declare that there is no conflict of interest.