Mosquitoes of North-Western Europe as Potential Vectors of Arboviruses: A Review

Background: The intensification of trade and travel is linked to the growing number of imported cases of dengue, chikungunya or Zika viruses into continental Europe and to the expansion of invasive mosquito species such as Aedes albopictus and Aedes japonicus. Local outbreaks have already occurred in several European countries. Very little information exists on the vector competence of native mosquitoes for arboviruses. As such, the vectorial status of the nine mosquito species largely established in North-Western Europe (Aedes cinereus and Aedes geminus, Aedes cantans, Aedes punctor, Aedes rusticus, Anopheles claviger s.s., Anopheles plumbeus, Coquillettidia richiardii, Culex pipiens s.l., and Culiseta annulata) remains mostly unknown. Objectives: To review the vector competence of both invasive and native mosquito populations found in North-Western Europe (i.e., France, Belgium, Germany, United Kingdom, Ireland, The Netherlands, Luxembourg and Switzerland) for dengue, chikungunya, Zika, West Nile and Usutu viruses. Methods: A bibliographical search with research strings addressing mosquito vector competence for considered countries was performed. Results: Out of 6357 results, 119 references were related to the vector competence of mosquitoes in Western Europe. Eight species appear to be competent for at least one virus. Conclusions: Aedes albopictus is responsible for the current outbreaks. The spread of Aedes albopictus and Aedes japonicus increases the risk of the autochthonous transmission of these viruses. Although native species could contribute to their transmission, more studies are still needed to assess that risk.


Introduction
Emerging or re-emerging disease outbreaks caused by arboviruses are increasing in Europe.Usually confined to tropical or subtropical countries, their risk of occurrence in Europe (especially in the countries of Western Europe) was considered as relatively low.In a context of global change, with an increase in travel, arboviruses can more easily expand outside of their historical range.Over the last two decades, arboviruses such as dengue, Zika or chikungunya achieved incursions in European countries [1][2][3], causing autochthonous human infections [4][5][6].Susceptible vertebrate hosts and competent vectors can interact, under appropriate environmental and climatic conditions, to cause outbreaks.Knowledge about the vector competence of native species and their distribution, however, remains limited.As such, overwintering thermophilic species are less likely to establish in northern European countries, as the annual isotherm becomes colder.At the present time, Aedes albopictus is considered as the vector species in autochthonous outbreaks of dengue and chikungunya in France [5].While the vector competence of invasive species is widely studied [7][8][9][10][11], only a few studies are available on native species [12].Competent native mosquitoes might have the potential to contribute to or to cause local outbreaks in addition to the risk related to invasive species [13].
In this work, we investigated the vector competence for dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), West Nile virus (WNV) and Usutu virus (USUV) according to the geographical distribution of mosquitoes in eight Western European countries (France-Corsica excluded, Belgium, Germany, United Kingdom, Ireland, The Netherlands, Luxembourg and Switzerland).

Materials and Methods
Our area of interest is defined as the "Cfb" climate of the Köppen-Geiger climate classification [14].Cfb defines a temperate oceanic climate (C) without a dry season (f) but with a warm summer (b) [15].Mainland France was investigated although the south of France is characterized as a "Csa" (temperate with a dry and hot summer) or "Csb" (temperate with a dry and warm summer) category.The island of Corsica categorized as "Csb" was excluded from our analysis [15].Mosquito inventories were then selected in our area of interest.The last European global inventory compiled by Schaffner et al. in 2001 [16] was used, completed with more recent national sources and finally compared with the last review available [17].For invasive species, updated European Center for Disease Prevention and Control (ECDC) maps were used (https://www.ecdc.europa.eu/en/disease-vectors/surveillance-anddisease-data/mosquito-maps).The vectorial status of mosquitoes was inferred from experimental data using infections in laboratory and/or viral detections in field-collected populations.
This review follows the PRISMA Journal Publishing protocol workflow [18]; the PubMed and Web of Science databases were screened from 11 January to 15 August 2019, with keywords presented on Supplementary File 1. Full-text articles in English language containing information on mosquito vector competence were selected.Articles in other languages containing critical information were included in the data set as "identified by other sources".The PRISMA flow chart is described in Figure 1.

Results
The compilation of mosquito inventories is summarized in Table 1.A detailed checklist is shown in Table 2, with the nine ubiquitous species highlighted in grey.Some species complexes (i.e., Aedes

Results
The compilation of mosquito inventories is summarized in Table 1.A detailed checklist is shown in Table 2, with the nine ubiquitous species highlighted in grey.Some species complexes (i.e., Aedes cinereus/geminus, Anopheles maculipennis complex, and Culex pipiens complex) were not identified at the species and/or biotype level.In these cases, the species denomination was annotated with an asterisk.
Detailed vector competence studies by species are presented in Table 3. Positive and negative results reported here are related to our area of study.Vector competence is defined as the ability of a mosquito to ingest, replicate and transmit a virus by biting.Firstly, species are considered to be competent if viral replication and detection of viral particles in saliva have been demonstrated.On the other hand, we do not consider the detection of viral RNA in mosquito pools as a relevant indication of vector competence.This information, however, may suggest an active virus circulation which could warrant further vector competence studies to identify potential vectors.
Results from experimental infections of European mosquito populations are listed in Table 4.
A Brief Summary of the Reviewed Arboviruses Is Presented Below

Chikungunya Virus
Chikungunya virus (genus Alphavirus, family Togaviridae) was reported for the first time in 1952-1953 in Tanzania [66].The original anthroponotic cycle allows the virus to circulate from Aedes mosquitoes to non-human primates.Outbreaks occur when an anthropophilic or opportunistic mosquito (e.g., Aedes aegypti, Ae. albopictus) completes the bridge between zoonotic and anthroponotic cycles.The girst reported outbreaks occurred in the southern hemisphere during the 1960s [67].Since then, three lineages have been identified: West-African, East-Central-South-African, and Asian lineages.During the 2000s, a new variant harboring a point mutation at the E1 glycoprotein gene (E1-A226V) was isolated during the outbreak in the islands of the Indian Ocean, making Ae.albopictus more efficient for the transmission of CHIKV [68].In Europe, the autochthonous transmission of CHIKV was first reported in Italy in 2007 [69].In our area of interest, the first autochthonous cases of CHIKV occurred in 2010 in Southern France [70].According to the ECDC, the six countries in this study reported 214 imported cases (Switzerland excluded) in 2012-2016 [71].An autochthonous circulation of CHIKV was observed in France 2014 and 2017 [4,70,72].An autochthonous outbreak of CHIKV also occurred in Italy in 2017 [73].
French Ae.albopictus and German Ae.japonicus are competent for CHIKV [11,45].Aedes detritus from the UK was found inefficient to transmit CHIKV [12].Aedes vexans from Northern Italy showed a low disseminated infection rate and its role in CHIKV transmission should not be neglected [13].In Europe, the declaration of imported cases of CHIKV shows three temporal peaks, namely February, April, and August to November (Figure 2) [71]-periods at which travelers return from endemic areas.While mosquitoes are absent in February, they become active in April; the dynamics of Ae. japonicus, Ae. koreicus and Ae.albopictus coincide with the peaks of imported cases promoting autochthonous transmission.
Viruses 2019, 11, x FOR PEER REVIEW 8 of 17 A Brief Summary of the Reviewed Arboviruses Is Presented Below

Chikungunya Virus
Chikungunya virus (genus Alphavirus, family Togaviridae) was reported for the first time in 1952-1953 in Tanzania [66].The original anthroponotic cycle allows the virus to circulate from Aedes mosquitoes to non-human primates.Outbreaks occur when an anthropophilic or opportunistic mosquito (e.g., Aedes aegypti, Ae. albopictus) completes the bridge between zoonotic and anthroponotic cycles.The girst reported outbreaks occurred in the southern hemisphere during the 1960s [67].Since then, three lineages have been identified: West-African, East-Central-South-African, and Asian lineages.During the 2000s, a new variant harboring a point mutation at the E1 glycoprotein gene (E1-A226V) was isolated during the outbreak in the islands of the Indian Ocean, making Ae.albopictus more efficient for the transmission of CHIKV [68].In Europe, the autochthonous transmission of CHIKV was first reported in Italy in 2007 [69].In our area of interest, the first autochthonous cases of CHIKV occurred in 2010 in Southern France [70].According to the ECDC, the six countries in this study reported 214 imported cases (Switzerland excluded) in 2012-2016 [71].An autochthonous circulation of CHIKV was observed in France 2014 and 2017 [4,70,72].An autochthonous outbreak of CHIKV also occurred in Italy in 2017 [73].
French Ae.albopictus and German Ae.japonicus are competent for CHIKV [11,45].Aedes detritus from the UK was found inefficient to transmit CHIKV [12].Aedes vexans from Northern Italy showed a low disseminated infection rate and its role in CHIKV transmission should not be neglected [13].In Europe, the declaration of imported cases of CHIKV shows three temporal peaks, namely February, April, and August to November (Figure 2) [71]-periods at which travelers return from endemic areas.While mosquitoes are absent in February, they become active in April; the dynamics of Ae. japonicus, Ae. koreicus and Ae.albopictus coincide with the peaks of imported cases promoting autochthonous transmission.

Dengue Virus
Dengue virus (genus Flavivirus, family Flaviviridae) includes four serotypes (DENV-1 to DENV-4).It is the most widespread arbovirus in the world and is reported in over 100 countries [76].Aedes aegypti and Ae.albopictus are the main vectors of DENV acting in urban cycles.According to the latest ECDC annual epidemiological report, covering the 2012-2016 period, 1562 cases (Switzerland excluded) were reported in our study area [74].The first autochthonous cases of dengue fever were reported in our area of interest (France) in 2010 [77].Other outbreaks in 2013 and 2015 also occurred in Southern France [5,78], where Ae. albopictus is well established and found in abundance [7].
According to the ECDC [74], the frequency of imported cases shows three peaks: January, from March to April, and from August to September (Figure 2).Mosquitoes are only active during the second and third peaks.

Zika Virus
Zika virus (genus Flavivirus, family Flaviviridae), was first isolated in the Zika forest of Uganda in 1947 [79].Since the Yap island epidemic in 2007, ZIKV has circulated on all continents except Europe [80].There are three main lineages: two from Africa and one from Asia [81].It is transmitted to humans (urban cycle) and primates (sylvatic cycle) by mosquitoes of the Aedes genus.In North-Western Europe, Ae. albopictus (France, Germany) and Ae.japonicus (Germany) are competent mosquitoes [8,37,46].Conversely, Culex pipiens s.l. and Cx.pipiens molestus are not competent for ZIKV [8].
According to the ECDC 2016 annual epidemiological report [75], ZIKV case reports are steadily increasing from January to reach a peak in August then decrease rapidly to end in October (Figure 2).European Ae. albopictus is competent to transmit ZIKV [8] and Ae.japonicus is poorly competent [37].

Usutu Virus
Usutu virus (genus Flavivirus, family Flaviviridae) was initially isolated in South Africa in 1959 [82].USUV was considered as exclusively transmitted in Africa until the first European outbreak occurred in Italy in 1996 [83].Since then, USUV emerged in five countries studied in this paper (Belgium, France, Germany, Switzerland, and The Netherlands) [84].USUV antibodies have been detected in resident and migratory birds in each country of our area of concern [85][86][87][88][89][90], except Ireland and Luxembourg.There are eight lineages of USUV.Five are European (European lineage 1-5) and three are African (African lineage 1-3) [84].The first human cases were reported in Africa in 1981 [91], and the first human neuroinvasive cases were reported in Italy in 2009 [92].USUV is transmitted in Europe by Cx. pipiens mosquitoes [51].The reservoir hosts of USUV are migratory and resident birds.USUV or antibodies have been detected in 58 bird species belonging to 26 families and 13 orders [93].Blackbirds (Turdus merula) seem to have the highest mortality rate among bird species affected by USUV [94].An exhaustive review about WNV and USUV has been published [93].Cx. pipiens s.l.from The Netherlands and the UK have been tested as competent for USUV strains, Bologna 2009 and SAAR-1776 [51,52].
While Cx. pipiens s.l. is considered the principal vector in temperate regions, Italian Ae. albopictus has been experimentally tested for USUV, with no clear-cut results [47].Ae. japonicus from Austria has been found positive to virus dissemination [95].To our knowledge, no other mosquito species of NW Europe has been demonstrated to be competent for USUV.

West Nile Virus
West Nile virus (genus Flavivirus, family Flaviviridae) was first recorded in the West Nile district of Uganda in 1937 [96].In natural conditions, it circulates between birds and bird-feeding mosquitoes.Mammals (mainly horses and humans) are considered dead-end hosts [97].In Europe, WNV is mainly transmitted by Culex mosquitoes.The first documented introduction of WNV in metropolitan France dates from 1962, in Camargue [98].The vector incriminated in 1964 was Culex modestus [99].During the following 40 years, WNV did not cause any human cases in France, and a low prevalence of antibodies was observed in human and equine populations during the 1970s [98].The resurgence of WNV in France occurred in 2000 with 76 laboratory confirmed equine cases.Sporadic detections of positive serology in humans and birds occurred during the following 15 years [88,100], until a new epidemic outbreak occurred in 2015 in southern France [101].Three years later, an outbreak occurred for the first time in Germany [102].The number of cases reported to the ECDC for 2018 was exceptionally high [103].
European mosquitoes display a variable susceptibility to WNV infection and transmission.Culex spp.from France, The Netherlands, Switzerland and Germany are competent for WNV lineages 1 and 2. Culex pipiens biotype pipiens, Culex pipiens biotype molestus and Culex torrentium from the studied area (France, The Netherlands, Switzerland and Germany) are competent for WNV lineage 1 and 2 [41,51,53,55,56,104]. Culex pipiens s.l.from Switzerland is susceptible to WNV infection but is not competent for WNV lineage 1 FIN Italy [55].The competence of Ae. detritus (United Kingdom) was demonstrated for WNV lineage 1 strain NY99 [12].
The vector competence of other field-collected species has not been successfully demonstrated for WNV: Ae. caspius (France) is susceptible to infection but not able to transmit [41].Ae. japonicus (Germany) could not be infected nor transmit WNV lineage 1 strain NY99 [38].

Discussion
North-Western Europe is at risk for emerging or re-emerging arboviruses.The epidemiology of arboviruses such as DENV, ZIKV or CHIKV is very different in Europe as compared to tropical or sub-tropical countries.In the latter countries, the existence of sylvatic cycles involving wild animals as reservoir hosts and arboreal canopy dwelling mosquitoes as vectors sustain a viral circulation of viruses all year long [105].Epidemics only occur when anthropo-zoophilic mosquitoes act as bridge vector for the transmission of the virus from animals to humans.In Europe, in the absence of any sylvatic cycle, autochthonous transmission is only caused when a competent vector becomes infectious after feeding on an imported human case.
The number of imported human cases of CHIKV, DENV and ZIKV peaks in January, March-April, and September-October [71,74,75], corresponding to vacations in Europe.The last two peaks may allow the launch of local transmissions.Regarding the January peak, there is no mosquito activity at that time in the considered area.Consequently, this peak will probably have no consequences regarding a local transmission.During the March-April peak, DENV and CHIKV can be transmitted by Ae. japonicus [23], and ZIKV by Ae. vexans if European mosquitoes appear to be competent (such as Canadian ones do) [30].The summer peak is highly correlated with the activity of Ae. albopictus.The competence of native populations has been characterized for CHIKV and ZIKV [8,45,46].Outbreaks have already occurred in southern European countries such as the occurrence of chikungunya in Italy [73] and dengue in France [5].Established vectors Ae. albopictus are also competent for transmitting ZIKV in Italy and Spain [106,107].
To date, Ae. albopictus and Ae.japonicus are the main species that could represent a risk of the transmission of the considered arboviruses in our area of interest.Indeed, native Ae.albopictus and Ae.japonicus can transmit CHIKV, DENV, ZIKV [11,37,45,46].This work highlights that invasive species represent the most probable candidates for the circulation of CHIKV, DENV, and ZIKV in our area of interest.To date, three species are established in our area of interest: Ae. albopictus, Ae. koreicus and Ae.japonicus.Ae. albopictus was first introduced in France in 1999 and has been established since 2004 [49].It is now widespread and abundant in the southern part of the country.It is also reported as established in the north-eastern part of France, in Germany, in Switzerland, in The Netherlands, and occasionally found in Belgium and the UK [108].Similarly, Ae. japonicus is well established in Belgium, France, Luxembourg, Germany, The Netherlands, and Switzerland [109,110].Ae. koreicus is implanted in Germany, Switzerland, and Belgium [111].While invasive species benefit from globalization (increased transportation of goods and people) to expand, native species are also experiencing ecological upheavals and contribute to increase the risk of arboviral emergences.Recently, An. plumbeus has switched from natural breeding sites to man-made sites [112] leading to consider its potential role as a vector for WNV [36].
While the vector competence of Ae. albopictus for the five viruses considered in this study has been largely examined, European native mosquitoes (Anopheles, Culiseta, Coquillettidia, Uranotaenia) were poorly investigated.The role of ornithophilic species, such as Cs.morsitans, and opportunistic species (mosquitoes that feed on mammalians as well as on birds or amphibians) like An. plumbeus or Ae.geniculatus, is still unknown, especially in the transmission of USUV and WNV.An. plumbeus, however, was tested competent for WNV, and Ae.geniculatus for CHIKV and WNV [35,36].
For USUV and WNV, the entanglement of mosquito populations with avian populations is necessary for sustaining the enzootic cycle.Recent years have shown a sharp increase in WNV cases [103].Although serological and molecular screening is regularly carried out in human [113,114] and avian populations [39,87,115], the screening of mosquito populations is less systematic.Also, WNV antibodies are more prevalent in migratory birds, while USUV are more prevalent in resident birds [115].USUV was more commonly found in mosquitoes than WNV [115].
Epizootics of WNV are episodic [98]; after the first emergence of WNV in France in 1962 [98], the virus was only detected again in the 2000s [116].Outbreaks were noted from 2004 to 2018 in mainland France [101,103,117], and in 2018 in Germany [102].The circulation of USUV was suspected in European birds in 2000-2005 [85,118].USUV emerged in Germany in 2011 [59], and then in France in 2015 [119].In 2016, an epizootic has globally affected France, Belgium, Germany and The Netherlands [84].The species involved in these outbreaks were probably members of the genus Culex (e.g., Cx. pipiens s.l.) [51,52].
These recent episodes recall our knowledge gaps on the vector competence of native and invasive species such as Ae.vexans, Ae. japonicus and other Aedes species for ZIKV, CHIKV and DENV; An. plumbeus, Ae. geniculatus, Cs. annulata, and Cx.torrentium for USUV and WNV.In the future, attention should be given to ubiquitous species which could be of importance if their vector competence happens to be demonstrated.

Table 2 .
Checklist of mosquitoes recorded per country.Species in grey rows were recorded in all the countries investigated in the study.X: species recorded; -: species not recorded; i: introduced species; species complexes for which final identification could not be achieved are labelled with an asterisk.

Table 3 .
Vector competence studies on European mosquito populations.Negative and positive experiments are written in normal and bold typeface, respectively.Studies related to our area of concern are indicated by a reference number.Studies related to experiments carried out outside of our area of concern are indicated with a reference number and an additional letter: E: Europe; W: elsewhere in the world.Since no capture of chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV) has been reported in our area of concern, these viruses are not mentioned in the field data part of the table.

Table 4 .
Experimental infections performed with North-Western European mosquito populations.